Intrahepatic spontaneous retrograde portal flow in patients with cirrhosis of the liver: reversal by food intake.

OBJECTIVE: The purpose of our study was to assess whether intrahepatic spontaneous retrograde portal flow in patients with cirrhosis of the liver can be reversed to a normal portal venous flow by food intake. CONCLUSION: Of the 18 cirrhotic patients with intrahepatic spontaneous retrograde portal flow, 16 (89%) showed a marked change in portal flow direction after food intake. This evidence strongly suggests that intrahepatic spontaneous retrograde portal flow may be reversible. Furthermore, this finding implies that regular food intake may be important in the maintenance of effective hepatic blood flow in cirrhotic patients.

An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p.

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.

Structural basis of diverse sequence-dependent target recognition by the 8 kDa dynein light chain.

Dyneins are multi-subunit molecular motors that translocate molecular cargoes along microtubules. Other than acting as an essential component of the dynein motor complex, the 89-residue subunit of dynein light chain (DLC8) also regulates a number of other biological events by binding to various proteins and enzymes. Currently known DLC8 targets include neuronal nitric oxide synthase; the proapoptotic Bcl-2 family member protein designated Bim; a Drosophila RNA localization protein Swallow, myosin V, neuronal scaffolding protein GKAP, and IkappaBalpha, an inhibitor of the NFkappaB transcription factor. The DLC8-binding domains of the various targets are confined within a short, continuous stretch of amino acid residues. However, these domains do not share any obvious sequence homology with each other. Here, the three-dimensional structures of DLC8 complexed with two peptides corresponding to the DLC8-binding domains of neuronal nitric oxide synthase and Bim, respectively, were determined by NMR spectroscopy. Although the two DLC8-binding peptides have entirely different amino acid sequences, both peptides bind to the protein with a remarkable similar conformation by engaging the symmetric DLC8 dimer through antiparallel beta-sheet augmentation via the beta2 strand of the protein. Structural comparison indicates that the two target peptides use different regions within the conformational flexible peptide-binding channels to achieve binding specificity. We have also re-determined the apo-form solution structure of DLC8 in this work. The structures of the DLC8/target peptide complexes, together with the dynamic properties of the protein, provide a molecular basis of DLC8's diverse amino acid sequence-dependent target recognition.

Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain.

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.

Formation of a native-like beta-hairpin finger structure of a peptide from the extended PDZ domain of neuronal nitric oxide synthase in aqueous solution.

Neuronal nitric oxide synthase (nNOS) is targeted to the cell membrane via interactions of its extended PDZ domain with PDZ domains of membrane-associated proteins including PSD-95 and alpha1-syntrophin. The formation of heterodimers between the nNOS PDZ domain and the PDZ domains of nNOS-binding proteins requires a stretch of continuous amino-acid residues C-terminal to the canonical nNOS PDZ domain. In this work, we show that a 27-residue peptide comprising the C-terminal extension of the extended nNOS PDZ domain is capable of binding to PSD-95. The structure of the 27-residue peptide in aqueous solution was determined using multidimensional NMR-spectroscopic techniques. The free peptide adopts a native-like beta-hairpin finger structure in aqueous solution. The results indicate that the C-terminal extension peptide of the nNOS PDZ domain may represent a relatively independent structural unit in the mediation of the interaction between nNOS and PDZ domain-containing proteins including PSD-95 and alpha1-syntrophin.

Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95.

The second PDZ domain of postsynaptic density-95 (PSD-95 PDZ2) plays a critical role in coupling N-methyl-D-aspartate receptors to neuronal nitric oxide synthase (nNOS). In this work, the solution structure of PSD-95 PDZ2 was determined to high resolution by NMR spectroscopy. The structure of PSD-95 PDZ2 was compared in detail with that of alpha1-syntrophin PDZ domain, as the PDZ domains share similar target interaction properties. The interaction of the PSD-95 PDZ2 with a carboxyl-terminal peptide derived from a cytoplasmic protein CAPON was studied by NMR titration experiments. Complex formation between PSD-95 PDZ2 and the nNOS PDZ was modelled on the basis of the crystal structure of the alpha1-syntrophin PDZ/nNOS PDZ dimer. We found that the prolonged loop connecting the betaB and betaC strands of PSD-95 PDZ2 is likely to play a role in both the binding of the carboxyl-terminal peptide and the nNOS beta-finger. Finally, the backbone dynamics of the PSD-95 PDZ2 in the absence of bound peptide were studied using a model-free approach. The "GLGF"-loop and the loop connecting alphaB and betaF of the protein display some degree of flexibility in solution. The rest of the protein is rigid and lacks detectable slow time-scale (microseconds to milliseconds) motions. In particular, the loop connecting betaB and betaC loop adopts a well-defined, rigid structure in solution. It appears that the loop adopts a pre-aligned conformation for the PDZ domain to interact with its targets.

Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide.

The PDZ domain of neuronal nitric oxide synthase (nNOS) functions as a scaffold for organizing the signal transduction complex of the enzyme. The NMR structure of a complex composed of the nNOS PDZ domain and an associated peptide suggests that a two-stranded beta-sheet C-terminal to the canonical PDZ domain may mediate its interaction with the PDZ domains of postsynaptic density-95 and alpha-syntrophin. The structure also provides the molecular basis of recognition of Asp-X-Val-COOH peptides by the nNOS PDZ domain. The role of the C-terminal extension in Asp-X-Val-COOH peptide binding is investigated. Additionally, NMR studies further show that the Asp-X-Val-COOH peptide and a C-terminal peptide from a novel cytosolic protein named CAPON bind to the same pocket of the nNOS PDZ domain.

Intermolecular contacts between the lambda-Cro repressor and the operator DNA characterized by nuclear magnetic resonance spectroscopy.

The specific interaction between lambda phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both 15N- and 13C/15N- labeled lambda-Cro in complex with unlabeled DNA, chemical shift assignments of the lambda-Cro-DNA complex were obtained using heteronuclear NMR experiments. Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock lambda-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of lambda-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecular NOEs were observed between the "helix-turn-helix" region comprising the alpha2 and alpha3 helices of the lambda-Cro protein and the major groove of the DNA. The methyl group of Thr17 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of lambda-Cro to several variants of the operator sequence.

Association between a focal spared area in the fatty liver and intrahepatic efferent blood flow from the gallbladder wall: evaluation with color Doppler sonography.

OBJECTIVE: The purpose of this study was to investigate whether fatty sparing adjacent to the gallbladder fossa is related to efferent blood flow from the gallbladder wall. SUBJECTS AND METHODS: Color and power Doppler sonography were performed in 80 patients with a fatty liver that appeared as a fine echogenic pattern with considerable deep attenuation on sonography. We analyzed whether color signal appeared around the spared area, the gallbladder wall, or both. Subsequently, when such signal was detected, pulse Doppler analysis of the signal was performed. RESULTS: Color signal indicating efferent blood flow from the gallbladder wall was detected in 25 (64%) of 39 patients with a focal spared area at the gallbladder fossa and in two (5%) of 41 patients without a focal spared area. This difference was statistically significant (p < .05). The waveform of efferent blood flow signals (n = 25) that were seen within spared areas was continuous in 23 (92%) of 25 patients and was pulsatile in the remaining two patients (8%). CONCLUSION: Blood flow from the gallbladder wall to areas of the spared liver was frequently revealed by color and power Doppler sonography; therefore, this blood flow may be associated with focal fatty sparing.

Protein inhibitor of neuronal nitric-oxide synthase, PIN, binds to a 17-amino acid residue fragment of the enzyme.

Neuronal nitric-oxide synthase (nNOS) is the primary nitric oxide (NO) regulator in neurons. The activity of the enzyme is inhibited by a protein inhibitor called PIN. We were able to purify large quantities of PIN overexpressed in bacterial cells. Analytical ultracentrifugation and chemical cross-linking studies showed that PIN exists as a monomer at low concentrations. The protein forms a high order aggregate at elevated concentrations. We have shown, using NMR spectroscopy, that the previously identified PIN-binding domain (PINB) of nNOS (residues 161-245) adopts a random coil structure in solution. By titrating 15N-labeled PINB with unlabeled PIN, the PIN-binding region of nNOS was precisely mapped to a 17-residue peptide fragment from Met-228 to His-244 of nNOS. NMR titration experiments also showed that PIN binds to nNOS with a 1:2 stoichiometry. A synthetic peptide corresponding to the identified PIN-binding region of nNOS was used to study the interaction between PIN and nNOS in detail. The functional implications of the results obtained from this study are discussed.

Solution structure of a protein inhibitor of neuronal nitric oxide synthase.

The structure of the neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), has been determined by NMR spectroscopy. Two N-terminal antiparallel alpha-helices pack against a four-stranded antiparallel beta-sheet in the C-terminal region of the protein, forming a two-layer alpha/beta plait. The three dimensional structure of PIN resembles the fold of the B-chain of aspartylglucosaminidase. A non-prolyl cis peptide bond was found between Pro 52 and Thr 53 of the protein. PIN has a large solvent-exposed hydrophobic surface that contains a cavity and is rimmed with positive charges. This surface may serve as the primary target-binding region for this multi-functional regulatory protein.

Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790.

Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211-218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-alpha induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-alpha-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123-234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.

Chronic encapsulated intracerebral hematoma associated with angiographically occult arteriovenous malformation--case report.

A 34-year-old female presented with a rare chronic encapsulated intracerebral hematoma associated with an angiographically occult arteriovenous malformation. A neovascularized fibrous capsule containing various stages of intracerebral hematoma formation was removed. These unusual entities mimic brain tumors or abscesses because of gradual growth and slowly progressive neurological deficits. Repeated bleeding or exudation from the capillaries of the capsule may allow expansion of the chronic encapsulated intracerebral hematoma.

Intrahepatic portosystemic venous shunt: diagnosis by color Doppler imaging.

Intrahepatic portosystemic venous shunt is a rare clinical entity; only 33 such cases have been reported. It may be congenital, or secondary to portal hypertension. Five patients with this disorder are presented, each of whom was diagnosed by color Doppler imaging, including waveform spectral analysis. One patient with clinical evidence of cirrhosis and portal hypertension had episodes of hepatic encephalopathy and elevated blood levels of ammonia. This patient had a large tubular shunt between the posterior branch of the portal vein and the inferior vena cava. Shunts of this type are considered to be collateral pathways which develop in the hepatic parenchyma as a result of portal hypertension. The other four patients had no evidence of liver disease, and all four evidenced an aneurysmal portohepatic venous shunt within the liver parenchyma. Shunts of this type are considered congenital. The diagnosis of intrahepatic portosystemic venous shunts was established by color Doppler imaging, which demonstrated a direct communication of color flow signals between the portal vein and hepatic vein, in addition to the characterization of the Doppler spectrum at each sampling point from a continuous waveform signal (portal vein) to a turbulent signal (aneurysmal cavity), and finally, to a biphasic waveform signal (hepatic vein). As demonstrated by the five patients, color Doppler imaging is useful in the diagnosis of an intrahepatic portosystemic hepatic venous shunt, and the measurement of shunt ratio may be useful in the follow-up and determining the therapeutic option.

Cervical intramedullary sarcoidosis.

Spinal cord involvement by sarcoid is quite rare. Only fourteen cases of histologically proven sarcoid without meningeal infiltration have been reported. We present the first CT scan description and the second description of the findings on MR imaging of intramedullary sarcoidosis. We also report successful surgical intervention in this case. In a review of surgical reports, we could find only 2 cases of improvement in symptoms after surgical intervention. Additionally, we stress the importance of early diagnosis using the various neuroradiological methods described. We feel that if surgery is performed at an early stage, it may be possible to obtain successful resection of the mass and a good clinical outcome.

Small hepatocellular carcinoma: diagnosis with US angiography with intraarterial CO2 microbubbles.

Ultrasonographic (US) angiography enhanced with intraarterial CO2 microbubbles, a contrast material used in US imaging, was performed of 103 histologically proved hepatocellular carcinomas (HCCs) smaller than 3 cm in diameter in 95 patients. The detection rate for hypervascular HCC with US angiography was compared with the rate of detection with conventional angiography, digital subtraction angiography (DSA), and computed tomography (CT) after intraarterial injection of iodized oil. Sensitivity in detection of hypervascular HCCs with US angiography was 86% (89 of 103 HCCs), compared with 63% (44 of 70 HCCs) detected with conventional angiography, 70% (23 of 33 HCCs) with DSA, and 82% (75 of 91 HCCs) with CT with iodized oil. US angiography depicted small hypervascular HCCs, especially those less than 1 cm in diameter, and helped clarify vascularity as isovascular or hypovascular in angiographically undetectable HCCs. Findings at US angiography assisted the choice of a therapeutic strategy for treatment of HCC, such as transarterial therapy, percutaneous ethanol injection therapy, or resection.