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The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell–cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.

The coxsackievirus and adenovirus receptor (CAR) is a cell surface protein that is proposed to be involved in cell-cell adhesion. Based on a yeast two-hybrid screen, co-immunoprecipitation and binding experiments, the intracellular tail of CAR was found to interact both in vivo and in vitro with the Ligand-of-Numb Protein-X2 (LNX2). The interacting domains between the two proteins were identified by truncation analyses and affinity chromatography. CAR and LNX2 protein expression in embryonic mouse tissues was analyzed by immunohistochemistry. The results suggest that CAR is a partner in a protein complex organized at specific subcellular sites by LNX2.

The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family.

Subsets of glycoproteins are thought to require lectin-like membrane receptors for efficient export out of the endoplasmic reticulum (ER). To identify new members related to two previously characterized intracellular lectins ERGIC-53/p58 and VIP36, we carried out an extensive database search using the conserved carbohydrate recognition domain (CRD) as a search string. A gene, more closely related to VIP36 than to ERGIC-53/p58, and hence called VIPL (VIP36-Like), was identified. VIPL has been conserved through evolution from zebra fish to man. The 2.4-kb VIPL mRNA was widely expressed to varying levels in different tissues. Using an antiserum prepared against the CRD, the 32-kDa VIPL protein was detected in various cell lines. The single N-linked glycan of VIPL remained endoglycosidase H-sensitive during a 2-h pulse-chase, even when the protein was overexpressed or mutated to allow export to the plasma membrane. VIPL localized primarily to the ER and partly to the Golgi complex. Like VIP36, the cytoplasmic tail of VIPL terminates in the sequence KRFY, a motif characteristic for proteins recycling between the ER and ERGIC/cis-Golgi. Mutating the retrograde transport signal KR to AA resulted in transport of VIPL to the cell surface. Finally, knock-down of VIPL mRNA using siRNA significantly slowed down the secretion of two glycoproteins (MR 35 and 250 kDa) to the medium, suggesting that VIPL may also function as an ER export receptor.

Hantavirus infections are a major public health concern worldwide. Their widespread geographical distribution and their ability to produce serious, often fatal, human disease underline the need for a system that allows manipulation of these viruses. We describe here the first successful establishment of a reverse genetics technology for Hantaan virus, the prototype of the genus Hantavirus. The system offers a unique opportunity to study the biology of hantaviruses, the pathogenesis of the diseases, and the efficacy of antiviral and prophylactic measures against hantavirus infections.

The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.

p58/ERGIC-53 is an animal calcium-dependent lectin that cycles between the endoplasmic reticulum (ER) and the Golgi complex and appears to act as a cargo receptor for a subset of soluble glycoproteins exported from the ER. We have determined the crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, to 1.46 A resolution. The fold and ligand binding site are most similar to those of leguminous lectins. The structure also resembles that of the CRD of the ER folding chaperone calnexin and the neurexins, a family of non-lectin proteins expressed on neurons. The CRD comprises one concave and one convex beta-sheet packed into a beta-sandwich. The ligand binding site resides in a negatively charged cleft formed by conserved residues. A large surface patch of conserved residues with a putative role in protein-protein interactions and oligomerization lies on the opposite side of the ligand binding site. Together with previous functional data, the structure defines a new and expanding class of calcium-dependent animal lectins and provides a starting point for the understanding of glycoprotein sorting between the ER and the Golgi.

The present paper proposes that trophic interplay among cells may represent the final common pathway for both genetic and environmental influences, and hence new criteria for the understanding of central nervous system (CNS) connectivity can be suggested. In particular, trophic signals may make up the common "language" through which genetic and epigenetic influences mold the CNS during development and the adult life. Furthermore, it will put forward the hypothesis that the developmental trophic interplay among cells leads to the formation of trophic units in the adult brain. A trophic unit is defined as the smallest set of cells, within the CNS, which act in a complementary way to support each other's trophism. The trophic units consist of neurons, glial cells, blood vessels, extracellular matrix (ECM). In particular, ECM gives support to the thin elongated cell processes and gives rise to selective chemical bridges between cell surfaces or between cell surfaces and the extracellular milieu. The trophic unit is a plastic device that not only assures neuronal survival, but also operates to adapt neuronal networks to new tasks by controlling extension of neuronal processes, synapse turnover and ECM characteristics. These plastic responses depend on the interplay of all the elements that constitute the trophic units. The concept of trophic unit may help to understand some features of neurodegenerative diseases, for example, the clustering of tangles in the neocortex and in the entorhinal cortex of Alzheimer's patients [corrected].

Members of the Bunyaviridae family acquire their envelopes by budding into the Golgi complex (GC). The accumulation of the membrane glycoproteins G1 and G2 in the GC probably determines the site of maturation. Here we have studied the intracellular transport and targeting to the GC of G1 and G2 of Uukuniemi virus, a member of the Phlebovirus genus, and report on their expression from cloned cDNAs either together or separately by using a T7 RNA polymerase-driven vaccinia virus expression system. When G1 and G2 were expressed together from a full-length cDNA as the p110 precursor, both proteins were localized to the Golgi complex, as evidenced by colocalization with the Golgi marker enzyme mannosidase II. Immunofluorescent staining indicated that G1 expressed alone also localized to the GC. However, pulse-chase experiments showed that G1 remained endoglycosidase H sensitive. G2 expressed alone remained associated with the endoplasmic reticulum (ER). G2 could be rescued from the ER and transported to the GC by coexpression with G1 from separate mRNAs. Coexpression also increased the efficiency of G1 transport to the GC. With none of the constructs could the glycoproteins be observed on the cell surface. These results show that efficient export of G1 and G2 from the ER requires coexpression of both proteins, in conformity with our previous results showing that G1 and G2 from heterodimeric complexes in the ER. Since G1 expressed alone is retained in the GC, we conclude that G1 contains a retention signal for localization to the GC. G2 might thus become associated with the GC indirectly via its interaction with G1.

Induction of basic fibroblast growth factor (bFGF) mRNA expression was studied in a Rose bengal induced focal cerebral ischemia during a time course of 2, 4, 24, 72 h and 7 days. Focal cerebral ischemia induced by Rose bengal resulted in a global upregulation in bFGF gene expression at the 24 h time-interval. This upregulation in bFGF gene expression was due to an upregulation in glial bFGF expression in most of the areas studied as seen by means of non-radioactive in situ hybridization in combination with immunocytochemistry for glial fibrillary acidic protein. However, in the piriform cortex a putative neuronal upregulation of bFGF could be detected by combination of non-radioactive in situ hybridization, immunohistochemistry for glial fibrillary acidic protein and nuclear staining with Neutral red. Semiquantitative data concerning bFGF mRNA expression were obtained by use of computer-assisted microdensitometry and revealed substantial increases in bFGF mRNA expression in the cingulate cortex, the neostriatum, a 1 mm marginal zone close to the external capsule and the olfactory tubercle at bregma levels 1 to 2 mm rostral to the lesion. No changes in bFGF gene expression were seen in field CA1 of Ammon's horn on the lesioned side and in dentate gyrus at bregma levels between -2.12 to -3.30 mm. We observed significant changes in bFGF upregulation in the caudate putamen, the piriform cortex and the amygdaloid region and the frontoparietal cortex at bregma levels -2.12 to -3.30 mm. These data indicate that photochemically induced focal cerebral ischemia leads to an early and global response in bFGF gene expression, which is due to an upregulation mainly in astrocytes. The observed widespread upregulation of the bFGF gene transcription rostral and caudal to the lesion is suggested to be due in part to neuronal glutaminergic connections between the areas investigated and in part due to increases in extracellular fluid signals (volume transmission).

To study the intracellular transport and targeting to the Golgi complex of the membrane glycoproteins G1 and G2 of Hantaan virus, we have expressed them together and separately using recombinant vaccinia viruses. When expressed from the same recombinant vaccinia virus, G1 and G2 were localized to the Golgi complex as analyzed by both immunofluorescence and subcellular fractionation. However, when the glycoproteins were expressed from separate recombinant viruses, both proteins remained in the endoplasmic reticulum. Using several monoclonal antibodies, it was found that G1 expressed alone did not acquire its correct conformation. Finally, if cells were coinfected with G1- and G2-expressing recombinant viruses, the proteins were again targeted to the Golgi complex. The N-linked glycans remained in all cases largely endoglycosidase-H sensitive. With none of the recombinant viruses were expression of the glycoproteins observed on the cell surface. Neither did chasing in the presence of cycloheximide result in the surface expression of G1 or G2. Our results indicate that for transport out of the endoplasmic reticulum and proper targeting to the Golgi complex, the two glycoproteins have to be coexpressed. The most likely interpretation is that G1 and G2 have to interact with each other in the endoplasmic reticulum in order to become transport competent.

A retrospective study was undertaken to assess whether stimulation of uterine contractility prior to surgical evacuation of a molar pregnancy will lead to an increased frequency of persistent trophoblastic disease. Forty-seven patients treated with chemotherapy for persistent trophoblastic disease after a hydatidiform mole between 1971 and 1988 were evaluated. The use of medical methods in this study group was compared to a control group of 219 patients with hydatidiform mole not requiring further treatment. A medical method, mainly treatment with prostaglandins, was used in 61.7% in the study group compared to 35.2% in the control group. This difference was, however, due to different stage distribution in the groups. Persistent disease was significantly correlated to uterine size and medical methods were mainly used in patients where uterine size corresponded to 15 weeks gestation or more. In this subset of patients, a medical method was used in the same frequency in both groups. Thus, large uterine size seems to be an independent risk factor. We conclude that stimulation of uterine contractility, which in Sweden is frequently used before surgical evacuation of the uterus in patients with hydatidiform mole and large uteri, carries no additional risk.

Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor.

One hundred and forty-seven women with uterine mixed Mullerian tumours (UMMT) treated at Radiumhemmet from 1936 through 1981 were reviewed. The prognostic value of clinical and histopathologic pata was analysed with bivariate and multivariate techniques. Stage, age and abdominal pain were found to be significant predictors of survival. Surgery and combined radiotherapy (intracavitary + external irradiation) gave in stage I, a lower local failure rate ( p = 0.006) and better overall survival ( p = 0.001) than surgery in combination with either intracavitary or external irradiation.

The 5′ terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of HeLa cells and is activated by Mg ++ . The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5′ terminal protein. The role of this enzyme in the uninfected cell is not known.

We have chemically synthesized an oligonucleotide 5'd(TGATTGATTGA)3' 3'd(ACTAACTAACT)5' that encodes the translation termination codon TGA in all three reading frames. After ligation of appropriate restriction endonuclease linkers to the ends, the double-stranded oligonucleotide (STOP-oligonucleotide) was joined to the plasmid pBR322 between the EcoRI and BamHI, or HindIII and BamHI sites, and the hybrid plasmids were transformed into Escherichia coli HB101. Four different constructions were obtained: (i) EcoRI-STOP-BamHI (STOP-oligonucleotide flanked by EcoRI and BamHI linkers; pKTH606), (ii) HindIII-STOP-BamHI (pKTH601), (iii) BamHI-STOP-HindIII (pKTH604), and (iv) HindIII-STOP-POTS-BamHI (two STOP-oligonucleotides in opposite orientation; pKTH605). The inserts in pKTH606 and pKTH601 were excised and transferred to a modified plasmid constructed previously for the expression and secretion of foreign gene products from Bacillus subtilis. The resulting secretion plasmids now contain the promoter/signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens joined to the STOP-oligonucleotide by EcoRI or HindIII linkers. Foreign genes can be cloned into these sites. The plasmids can be used to express foreign genes truncated at their C-terminal end and therefore lacking their own translation termination codon. One such plasmid has been successfully used to express the Semliki Forest virus (SFV) membrane protein E1 truncated at its C-terminus.

We have determined the complete nucleotide sequence of the virion M RNA segment of Uukuniemi virus (Uukuvirus genus, Bunyaviridae) from cloned cDNA. The RNA that encodes the two membrane glycoproteins G1 and G2 is 3231 residues long (mol wt 1.1 × 106). The 5′ and 3′ ends of the RNA are partially complementary to each other for some 30 bp, enabling the formation of a stable panhandle structure (ΔG = −40 kcal/mol) and the circularization of the molecule. The extreme 5′ and 3′ terminal nucleotides are identical for 10 to 13 residues to those of the M RNA of Punta Toro and Rift Valley fever viruses, two members of the Phlebovirus genus. A single open reading frame comprising 1008 amino acid residues (mol wt 113,588) was found in the mRNA-sense strand between nucleotides 18 and 3042. This probably corresponds to the previously identified 110,000-Da precursor (pl 10) of G1 and G2. By comparing the partial aminoterminal sequences of purified G1 and G2 with the deduced protein sequence we confirmed that the gene order is NH2-G1-G2-COOH. Both mature G1 and G2 are preceded by a stretch of 17 predominantly hydrophobic amino acids likely to represent the signal sequences. At their COON-terminal ends, G1 and G2 have a hydrophobic stretch of amino acids, 19 and 27 residues, respectively, that probably anchors the proteins to the lipid bilayer. The sequence indicates that mature G2 is 495 amino acids long (mol wt 54,869), whereas the exact size of G1 is unclear, since the location of the COON-terminus of G1 is not known. An upper value of 479 amino acids (mol wt 55,181) can, however, be suggested. Both G1 and G2 contain four potential glycosylation sites for Asn-linked glycans and both are unusually rich in cysteines, 6.1% in G1 and 5.4% in G2. Comparison of the amino acid sequence of the M RNA product of Uukuniemi virus with that of Punta Toro and Rift Valley fever viruses showed in both cases a weak homology that was more pronounced for the proteins located at the COON-terminal end of the precursor. This suggests a distant evolutionary relationship between the Phlebo- and Uukuvirus genera.

The gene coding for the Semliki Forest virus (SFV) membrane protein El was joined to a secretion vector containing the promoter and signal sequence regions of the α-amylase gene from Bacillus amyloliquefaciens. To facilitate secretion, the regions coding for the N-terminal signal peptide (the 6K protein) and the C-terminal hydrophobic transmembrane domain of the El gene were deleted. After transformation into B. subtilis, E1 was shown by immunoblotting to be expressed at a low level (about 0.5–1 mg/1). Contrary to what was expected, most of the E1 remained cell-associated. Deletion of a residual 7 C-terminal amino acids from the 6K region neither increased the level of expression nor significantly improved the secretion. Immunofluorescence microscopy of protoplasts prepared from B. subtilis cells expressing E1 suggested that the cell-associated E1 was located at the outer surface of the bacterial membrane. Addition of protease inhibitors to the culture medium somewhat increased the amount of extracellular E1, suggesting that proteolytic degradation of the foreign gene product may be one reason for the low level of expression. This conclusion was also supported by experiments carried out in Bacillus minicells, which indicated that the expression of the E1 gene in the absence of synthesis of bacterial proteases was about the same as that of α-amylase expressed from the cloned gene using the same promoter and signal sequence.

A randomised controlled trial to investigate the efficacy of mass screening with single-view mammography in reducing mortality from breast cancer was started in Sweden in 1977. 162 981 women aged 40 years or more and living in the counties of Kopparberg and Ostergotland were enrolled in the study and divided at random into 2 groups. Each woman in the study group was offered screening every 2 or 3 years depending on age. Women in the control group were not offered screening. This report is confined to the 134 867 women aged 40-74 years at date of entry. The results to the end of 1984 show a 31% reduction in mortality from breast cancer and a 25% reduction in the rate of stage II or more advanced breast cancers in the group invited to screening. 7 years after the start of the study the excess of stage I cancers in the study group largely outweighs the deficit of advanced cancers.

Semliki Forest virus (SFV) was transferred serially with undiluted inoculum for 24 successive passages in BHK cells and the virus-specific RNAs were isolated from infected cells and from virus particles released into the medium. The infectivity and hemagglutination titers started to drop after 4 serial passages. A new virus-specific intracellular RNA species sedimenting at 18 S appeared in the 4th passage and remained the dominant DI-RNA up to the 17th passage. The 18 S RNA population was incorporated very inefficiently into viral particles, but was replicated more rapidly than other viral RNAs. Another DI-RNA, sedimenting at 24 S, was found in virus particles starting at the 4th passage. The 24 S RNA replicated less efficiently but was incorporated in virus particles 3 to 5 times more efficiently than the 18 S RNAs. After 18 undiluted passages a switch in the distribution of intracellular RNAs resulted in the predominance of a 24 S RNA species. The early and late 24 S RNA species were found to be related but not identical when compared by ribonuclease Tl oligonucleotide mapping. The results indicated that the early 24 S RNA fingerprint closely resembled that of the 18 S DI-RNA population, whereas the late 24 S RNA had a lower complexity. A 33 S DI-RNA species isolated from virus particles at the 21st passage also had a simple T1 oligonucleotide pattern indistinguishable from that of the late 24 S RNA. These results suggest that the late 24 S DI-RNA molecules contain multiple repeated sequences similar to those recently found in the 18 S DI-RNA.

Sucrose gradient centrifugation of SDS-treated, purified Uukuniemi virus labeled with 32 P , gave three reproducible peaks of single-stranded RNA, which sedimented at 27–29 S (L), 22 S (M), and 17–19 S (S). In polyacrylamide-agarose gels these RNAs separated into four peaks with apparent molecular weights of about 4.1 × 106 (L), 1.9 × 106 (M), 0.88 × 106 (S1), and 0.78 × 106 (S2), respectively. The major M and L RNAs, which represented about 75% of the total, were found in a constant molar ratio of 1:4.6. Treatment of the RNA species with 1 M formaldehyde or by heating to 100 ° did not result in a degradation of the larger to the smaller RNAs. The base compositions of the RNA species did not differ significantly from each other, but were clearly different from those of the ribosomal 28 S and 18 S RNAs extracted from host chick embryo cells. RNAs with identical sedimentation values and mobilities in gels were found from Uukuniemi virus-infected cells labeled with [ 3 H ]uridine in the presence of actinomycin D.

Uukuniemi virus, the prototype of a new serological group of arboviruses, was grown in BHK21 cells and purified by isopycnic centrifugation in sucrose gradients. The purified virus had a sedimentation constant of about 450 S. The buoyant density of the virus was 1.20 g/cm 3 in CsCl and 1.18 g/cm 3 in sucrose. Sucrose gradient centrifugation of SDS-treated virus gave two RNA peaks (21 and 27 S). Two polypeptides of molecular weight 65,000–75,000 and about 25,000 could be separated from whole virus by SDS-disc electrophoresis. Two fractions were obtained by CsCl gradient centrifugation of Nonidet-treated virus. A fraction with a density of less than 1.25 g/cm 3 contained phospholipids and the larger protein (molecular weight 65,000–75,000). This fraction apparently contains disrupted virus envelopes. The other fraction with a density of 1.31 g/cm 3 contained RNA and the smaller protein (molecular weight 25,000). Electron micrographs of the latter fraction showed strands about 2.5 nm thick and over 1000 nm long, apparently uncoiled nucleoprotein of the virus. The mode of packing of the nucleoprotein within the virion is still obscure.