CT appearance of persistent left superior vena cava, anomalous right superior pulmonary venous return into the right-sided superior vena cava and a sinus venosus-type atrial septal defect.

We present findings of persistent left superior vena cava, anomalous right superior pulmonary venous return into the right-sided superior vena cava and a sinus venosus-type atrial septal defect detected incidentally by CT pulmonary angiography. To our knowledge, there has been no previous case report with all of the above findings detected by CT. In addition to the radiological findings and their clinical significance, the anatomy and embryological explanation of each anomaly is discussed.

A large teratoma of the hard palate: a case report.

Congenital teratoma is a rare malformation, and few papers have been published about it. We present a large teratoma that arose from the hard palate in a neonate. The obstructive mass caused maternal polyhydramnios and was identified prenatally by ultrasonography. The mother went into labour at 35 week's gestation at home. The child was in respiratory distress as a result of airway obstruction, and a tracheostomy was done when she was 4hours old. She also had major cardiac abnormalities. The palatal mass was removed successfully at 4 weeks of age. The typical components of a teratoma were identified including immature neural glial tissue.

A survey of demand for specialist restorative dental services.

OBJECTIVES: To determine the level of demand from general dental practitioners (GDPs) for specialist restorative dental services in the Yorkshire region. To investigate barriers and promoters to referral and GDPs' perceptions of restorative mono-specialists. METHODS: A postal questionnaire was sent to 301 randomly selected GDPs (stratified for location) registered in the six Family Health Service Units in Yorkshire. Questionnaires were piloted prior to release; reminders were sent to non-respondents. RESULTS: A response rate of 72% (n=217) was achieved, of these 195 questionnaires were useable (65% useable response rate). Results showed a large demand for restorative specialist services. Main promoters for National Health Service (NHS) referral were dentolegal issues (77% GDPs ranked this as a top three promoter) and for private referral increasing patient expectations (78%). The top barriers against referral were length of waiting lists for NHS patients (79% GDPs ranked this as a top three barrier) and high cost of treatment for private patients (88%). Excessive distance to specialist centre was the greatest barrier common to both NHS and private referrals. Fifty-eight per cent of GDPs would prefer to refer private patients to mono-specialists, compared with 5% who would prefer restorative specialists. CONCLUSIONS: There is a strong demand for specialist restorative services which may increase in the future. Results indicate a high regard for mono-specialists. Overall demand is for a prompt, locally based, low cost referral service.

Phage display for target-based antibacterial drug discovery.

Increasing bacterial drug resistance and hard-to-eradicate opportunistic infections have created a need for new antibiotics. Sequencing of microbial genomes has yielded many new potential targets for antibacterial drug discovery. However, little is known about the biochemical activities of many of these targets, making it difficult to develop HTS assays for them. Peptides isolated by phage display can be used as 'surrogate ligands' in competition assays for screening of targets of unknown function with small-molecule libraries. These screening assays can be adapted into a variety of high-throughput formats, including those based on radioactive, luminescence or fluorescence detection.

Definition of a consensus transportin-specific nucleocytoplasmic transport signal.

The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.

Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein.

The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin alpha/importin alpha, which acts as the NLS receptor, and karyopherin beta1/importin beta, which binds karyopherin alpha and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin beta1, termed karyopherin beta2 or transportin, and does not require a karyopherin alpha-like adapter protein. A yeast homolog of karyopherin beta2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin beta1, but not the Kap104p homolog karyopherin beta2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin alpha. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta.

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.

A nuclear role for the Fragile X mental retardation protein.

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA-binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV-1). Mutation of this FMRP NES results in mis-localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev-dependent nuclear RNA export and specifically binds a nucleoporin-like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV-1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay.

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.

Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection.

The nef gene product encoded by the mac239 proviral clone of simian immunodeficiency virus (SIV) markedly enhances viral replication and pathogenesis in vivo. We have used this biologically active nef isolate to examine the phenotype of Nef in retrovirally transduced human T cells in culture. SIV Nef is shown to dramatically inhibit cell-surface expression of the CD4 glycoprotein without significantly affecting the total steady-state level of cellular CD4. This downregulation of the cell-surface CD4 receptor for human immunodeficiency virus type 1 (HIV-1) infection correlated with the acquisition of resistance to superinfection by HIV-1. However, SIV Nef did not affect the level of gene expression directed by the HIV-1 long terminal repeat. It is hypothesized that downregulation of cell-surface CD4 by Nef facilitates the efficient release of infectious progeny virions and, hence, viral spread in vivo.

A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor.

A modified double-sandwich enzyme-linked immunosorbent assay (ELISA), which was developed for human and canine von Willebrand's factor (vWF), was adapted for quantitation of vWF in other species. In addition to human and dog plasmas, 12 other mammalian plasmas that were surveyed exhibited significant cross-species reactivity with antibodies specific for canine vWF or monoclonal antibodies against porcine or bovine vWF. Mixed combinations of monoclonal antibodies and various polyclonal antibodies were also used as sandwich or capture antibodies. The ability of this multispecies ELISA to detect less than 0.002 U of vWF per milliliter of plasma in a large number of species enhances its utility for both research and clinical diagnostic applications. A quantitative assay for rabbit vWF, which exhibits poor cross-species reactivity with most antibodies, was constructed with anti-porcine monoclonal antibody W1-5 and goat-anti-canine vWF as capture and sandwich antibodies, respectively. The same conjugate antibody configuration was used to visualize rabbit vWF multimers by immunoblotting. Specificity of the assay for vWF in human, dog, pig, and horse plasmas was confirmed by use of species-specific vWF-deficient plasmas. In other species, for which vWD plasmas were not available, ELISA specificity for vWF was demonstrated by recovery of greater than 75% of the ELISA-reactive antigen coincident with vWF multimers in the high-molecular-weight (greater than 500 kd) fractions of purified plasmas. This multispecies ELISA permits, for the first time, the measurement of vWF in a variety of mammals for which species-specific immunologic reagents do not currently exist. The results also suggest that certain vWF epitopes have been highly conserved among phylogenetically diverse species.

A sensitive immunoassay for von Willebrand factor.

We have developed an ELISA specific for canine von Willebrand factor antigen (vWF:Ag) that also strongly reacts with the VWF:Ag of humans and many other vertebrates. This assay was designed to avoid the use of immunoreagents of human origin, however, commercially available antibodies to human vWF:Ag may also be used. von Willebrand factor (vWF) was quantitated using a modified double-sandwich ELISA with polyclonal antibodies specific for canine vWF:Ag. The assay was as sensitive for measuring canine vWF:Ag as previously published immuno-radiometric assays and the most sensitive ELISA for human vWF:Ag. Employing commercially available antibodies to human vWF:Ag in the same double-sandwich configuration, the lower limit of detection for human vWF:Ag was 4.8 x 10(-6) units/ml, lower by a factor of ten than previously reported ELISAs. In addition, a wide range of vWF:Ag levels can be determined with just a single plasma dilution. The assay readily distinguishes type III von Willebrand disease from other types of von Willebrand disease having very low levels of vWF. This vWF ELISA can be used to evaluate large numbers of plasma samples simultaneously and is therefore well-suited for large-scale screening programs.

Bleeding disorder (von Willebrand disease) in a quarter horse.

Bleeding diathesis in a Quarter Horse filly was caused by von Willebrand disease. Hemorrhage occurred mainly from mucosal surfaces and after trauma. Quantitative and qualitative measurements of plasma von Willebrand factor (vWF) documented a specific deficiency of vWF high molecular weight multimers, and concurrently greater than expected deficiency of vWF activity relative to vWF concentration. These findings are characteristic of type-II von Willebrand disease in human beings. Application of vWF assays used in human and small animal medicine now permits evaluation of vWF and diagnosis of von Willebrand disease in horses with bleeding disorders.

Conformational changes in von Willebrand's factor protein: effects on the binding of factor VIII-coagulant.

The effects of conformational changes in purified canine von Willebrand's factor (VWF) were investigated to explore the relationship between its factor VIII-related antigen (VIII:AG) and ristocetin cofactor (RCoF) properties and the factor VIII-coagulant (VIII:C) binding site(s). Binding of VIII:C from canine von Willebrand's disease (VWD) plasma by VWF was used to measure the combining reaction of these proteins. The VWF was denatured to varying degrees by exposure to temperature and pH extremes, low ionic strength, and 6 M urea. Various treatments resulted in three types of change: elimination of RCoF, VIIIR:Ag, and VIII:C binding, removal of RCoF activity alone; or elimination of RCoF and retarded elution of VIII:Ag and VIII:C. As long as VIIIR:Ag reactivity was maintained, binding of VIII:C could be demonstrated; but in the absence of VIIIR:Ag, neither RCoF activity nor VIII:C binding remained. These results suggest that VIII:C binding and RCoF sites are separate on VWF and that interaction between VIII:C and VWF is possible even after significant structural changes occur in VWF. Furthermore, RCoF is more vulnerable to denaturation than the antigenic site. The spectrum of conformational changes that affect the properties of VWF may parallel the various recognized subtypes of VWD.

Assessing the specificity of anti-canine-factor VIII-related antigen.

Antibodies to canine factor VIII-related antigen should be monospecific to accurately study this protein's location in tissue and its structure. Because no uniformly accepted standard for monospecificity exists for anti-factor VIII-related antigen, we have compared the sensitivities of three common evaluation techniques--immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis. Unabsorbed rabbit anti-canine factor VIII-related antigen appeared monospecific against whole plasma by all three methods; however, multiple specificities were recognized against a plasma cryoconcentrate. These results demonstrate the insensitivity of the techniques under certain conditions, which can lead to erroneous interpretation of data.

A practical technique for preparation of antiserum to canine factor VIII-related antigen.

Identification and measurement of canine factor VIII-related antigen (VIIIR:Ag) has many clinical and research applications, including differential diagnosis of hemophilia A and von Willebrand's disease, use as a marker for specific cell types, and elucidation of the structure of this factor VIII component. We have developed a practical method for producing antibodies to canine VIIIR:Ag that uses 2% agarose filtration for purification and identifies the antigen by correlation with the elution of the peak A280 fraction in the void volume. Antisera for electroimmunoassay (EIA) can be produced in less than four weeks from simple starting materials and with commonly available laboratory equipment. This technique would be useful for either clinical veterinary or comparative research laboratories.

Efficiency and precision of electroimmunoassay for canine factor VIII-related antigen.

Factors that improved the efficiency, precision, and sensitivity of the electroimmunoassay for canine factor VIII-related antigen were investigated. These included rapid electrophoresis, increased rocket heights, deletion of the gel-washing step, and doubling of the sample capacity per plate. The modified assay was reliable and accurate, could be completed within 3 hours, and permitted twice as many determinations. Other experimental variables that affected the assay were the type of agarose and sample diluent, storage conditions of samples before assay, source of antibody, and use of 2 mM calcium lactate.

Binding of low-molecular-weight canine factor VIII coagulant from von Willebrand plasma to canine factor VIII-related antigen.

Plasmas having no detectable factor VIII-related antigen but moderate factor VIII coagulant were obtained from two unrelated dogs homozygous for von Willebrand's disease and with the severe clinical expression of the disease. when these plasmas were gel-filtered in a buffer at physiologic ionic strength, the factor VIII coagulant eluted in the bed volume as a single well-defined peak. Addition of protease inhibitors, including diisopropylfluorophosphate, did not change the elution pattern. Each plasma was then combined individually with plasmas from six different mutants of canine haemophilia, all of which had normal factor VIII-related antigen but no detectable factor VIII coagulant. The factor VIII coagulant elution profile of these combined plasma resembled that of normal canine plasma. Slightly over half of the recovered factor VIII coagulant coeluted in the void volume with the factor VIII-related antigen; the rest eluted as a second, distinct peak of lower molecular weight. These results demonstrated that part of the factor VIII coagulant of the von Willebrand plasmas had bound to the factor VIII-related antigen of the haemophilic plasmas. This finding supports the theory that factor VIII exists as a macromolecular complex of nonidentical components in normal citrated plasma.