Short class I major histocompatibility complex cytoplasmic tails differing in charge detect arbiters of lateral diffusion in the plasma membrane.

Directed and Brownian movement of class I major histocompatibility complex (MHC) molecules on cell membranes is implicated in antigen presentation. Previous studies indicated that the class I MHC cytoplasmic tail imposes constraints on the molecule's diffusion. Here we used single particle tracking to study the mobility of the wild-type mouse H-2L(d) class I MHC molecule and of seven cytoplasmic tail variants. Six of the variants have cytoplasmic tails of four or seven residues (differing in net charge), and one is tailless, yet all are susceptible to confinement in membrane domains. However, truncation of the cytoplasmic tail to 0-4 residues decreases the proportion of particles exhibiting confined diffusion and increases the proportion exhibiting simple diffusion. Particularly for the truncated mutants (tail length of 0-7 residues), many of the particles have complex trajectories and do not move at a constant speed or in the same mode of diffusion throughout the observation period. Several particles of the tailless H-2L(d) mutant display a type of directed diffusion that is rarely observed for other H-2L(d) mutants. Taken together, these data show that even short cytoplasmic tails can influence markedly class I MHC mobility and that cytoplasmic tail length and sequence affect the molecule's diffusion in the membrane.

Phosphorylation of class I MHC molecules in the absence of phorbol esters is an intracellular event and may be characteristic of trafficking molecules.

Class I Major Histocompatibility Complex (MHC) molecules are displayed at the cell surface where they present antigenic peptides to T lymphocytes. Class I MHC molecules undergo cytoplasmic domain phosphorylation on a serine residue late in their biosynthesis. Here we show that phosphorylation occurs on mature, beta(2)-microglobulin-associated class I MHC molecules in a mouse lymphoid cell line. Both recently synthesized class I MHC molecules and molecules which are at least 3 h old become phosphorylated. Approximately 14% of phosphorylated class I MHC molecules occur at the cell surface. Density gradient analysis indicates that phosphorylated class I MHC molecules also occur in lamp(+) intracellular compartments and in fractions containing rab4, a GTP-binding protein associated with recycling endosomes. Class I MHC molecules are endocytosed and recycled to the cell surface in these cells. Furthermore, the lysosomotropic drug, primaquine, inhibits both class I MHC phosphorylation and its recycling back to the cell surface, suggesting that phosphorylation is related to class I MHC recycling. These observations are intriguing since several studies have shown that class I MHC molecules can acquire antigenic peptides in NH(4)Cl-sensitive compartments. Hence, class I MHC phosphorylation may play a role in regulating intracellular sorting through these compartments.

Magnetic resonance imaging in internal carotid artery agenesis with computed tomography and angiographic correlation--case reports.

The authors present two cases of agenesis of the internal carotid artery (ICA) discovered incidentally on magnetic resonance imaging and confirmed on computed tomography, magnetic resonance angiography, and conventional angiography. They also propose a clinical algorithm for the workup of patients with suspected absence of the ICA.

Thoracopancreatic fistula: clinical and imaging findings.

PURPOSE: The goal of this work was to describe the clinical and imaging features of thoracopancreatic fistula, a rare complication of pancreatitis. METHOD: Nine cases of thoracopancreatic fistula proved by thoracentesis, endoscopic retrograde cholangiopancreatography (ERCP), or surgery were retrospectively and independently reviewed by two abdominal radiologists. All available imaging examinations [chest radiographs = 9, CT = 9, MR and MR cholangiopancreatography (MRCP) = 2, and ERCP = 6] were analyzed, and findings were recorded on a standardized datasheet. Available medical records (n = 7) were reviewed to determine the clinical presentation of the patients and thoracentesis results. RESULTS: Seven of the nine patients presented with pulmonary symptoms such as dyspnea or cough. Of the seven patients with pleural fluid analysis, all demonstrated elevated amylase levels (mean 13,007 U/L). Imaging examinations revealed pancreaticopleural fistulas in six patients, a mediastinal pseudocyst in one patient, and both a pancreaticopleural fistula and a mediastinal pseudocyst in two patients. Chest radiography showed pleural fluid collections in eight patients. CT demonstrated a fluid-containing fistula in all nine patients. MR and MRCP depicted a fistula extending from the abdomen to the pleural space in the two patients with MR correlation. ERCP showed pancreatic ductal changes characteristic of chronic pancreatitis in the six patients with ERCP correlation but failed to demonstrate the fistula in two of the six patients. CONCLUSION: The CT, MR, MRCP, or ERCP finding of a fluid-filled tract extending from the pancreas to the thorax is characteristic of a thoracopancreatic fistula, particularly when identified in a patient who presents with pulmonary symptoms and a history of chronic pancreatitis.

MR cholangiography: technical advances and clinical applications.

Magnetic resonance (MR) cholangiography is a fast, accurate, noninvasive alternative to endoscopic retrograde cholangiography (ERC) in the evaluation of biliary tract disease. Technical improvements in imaging sequences (eg, half-Fourier rapid acquisition with relaxation enhancement) and use of phased-array coils allow high-quality imaging comparable to that available with ERC. In choledocholithiasis, common bile duct stones as small as 2 mm can be detected with MR cholangiography and appear as low-signal-intensity foci within the high-signal-intensity bile. MR cholangiography may help establish the diagnosis of malignant obstruction and is useful in the evaluation of patients in whom ERC was unsuccessful or incomplete. The role of MR cholangiography in the evaluation of intrahepatic duct disease continues to evolve. MR cholangiography plays a crucial role in evaluating postsurgical biliary tract alterations and can be used to demonstrate a variety of congenital anomalies of the biliary tract (eg, aberrant ducts, choledochal cysts, pancreas divisum). In addition, intentional or incidental imaging of the gallbladder with MR cholangiography can be used to identify calculi or help determine the presence and extent of neoplastic disease.

Half-Fourier RARE MR cholangiopancreatography: experience in 300 subjects.

PURPOSE: To determine prospectively the clinical applications and diagnostic accuracy of half-Fourier rapid acquisition with relaxation enhancement (RARE) magnetic resonance (MR) cholangiopancreatography (MRCP) in a large patient population. MATERIALS AND METHODS: Breath-hold, heavily T2-weighted half-Fourier RARE MRCP was performed in 265 patients with suspected pancreaticobiliary disease and in 35 control patients without symptoms or signs referrable to the biliary tract or pancreatic duct. MRCP findings were correlated with those at direct cholangiography, pathologic examination, cross-sectional imaging, and clinical follow-up. RESULTS: Diagnostic MRCP examinations were obtained in 299 (99.7%) subjects. MRCP yielded an accuracy of 100% in determining the presence of pancreaticobiliary disease, the presence and level of biliary obstruction, and obstruction due to bile duct calculi. The accuracy of MRCP and MR imaging in determining the presence and level of malignant obstruction was 98.2%. MRCP obviated endoscopic retrograde cholangiopancreatography (ERCP) by excluding choledocholithiasis in patients with acute pancreatitis (n = 13) and nonspecific abdominal pain (n = 82). In patients with sclerosing cholangitis and acquired immunodeficiency syndrome cholangiopathy, MRCP depicted the biliary tract as clearly as did ERCP (n = 9). After failed ERCP, MRCP delineated the pancreaticobiliary tract and helped determine therapeutic options (n = 27). CONCLUSION: Half-Fourier RARE MRCP enables accurate evaluation of pancreaticobiliary disease and obviates ERCP in some patients.

Intraoperative monitoring and postoperative imaging of hepatic cryosurgery.

Because intraoperative sonography displays segmental anatomy, allows discovery of more lesions than previously suspected from preoperative imaging, surgical inspection, or palpation, and permits more certain diagnosis of problematic masses, it facilitates surgical decision-making when liver resection or cryoablation is anticipated. Intraoperative sonography provides a guidance modality to accurately place cryosurgery probes in liver masses. More precise treatment of metastatic and primary tumors of the liver is possible with cryoablation because intraoperative sonography provides a means of monitoring the growth of the enlarging freeze zone to insure adequate surgical margins. Postoperative detection of acute complications after cryosurgery is best done with computed tomography. Normally cryosurgery defects shrink with time and lose the peripheral contrast opacification seen after surgery. Gas collections, seen as a result of tissue necrosis, must be discriminated from infection. Tumor recurrence can be detected well with computed tomography or magnetic resonance imaging following hepatic cryosurgery.

Imaging features of radiation-induced changes in the abdomen.

After external-beam radiation therapy, radiation-induced changes may be observed in abdominal and pelvic organs at imaging. In the liver, an area of low attenuation corresponding to the radiation port (or an area of hyperattenuation if the underlying liver tissue shows fatty change) can be seen at computed tomography (CT) performed within 3-6 months after therapy. Later, the liver may be fibrotic and contracted. In the stomach, small intestine, and colon, wall thickening and edema are early manifestations. Ulcers may also be observed. Long-term complications include strictures and fistulas. After irradiation of the kidneys, altered attenuation of the renal parenchyma may be seen at CT. Ureteral strictures, typically involving the distal ureter, may be observed after pelvic irradiation. The bladder may be small and contracted with a thickened wall after radiation exposure. Fistulas between the bladder and other pelvic organs sometimes occur. Typical musculoskeletal changes include growth abnormalities in skeletally immature patients, fatty replacement of bone marrow, and radiation osteitis. Radiation-induced neoplasms are also recognized after therapy.

Renal lymphoma mimicking clear cell sarcoma in a pediatric patient.

Lymphoma involving the kidneys typically presents as bilateral renal enlargement with multiple nodules [1-6]. Solitary masses, diffuse parenchymal infiltration, or engulfment of the entire kidney by diffuse disease may occur but are more rare. We report the case of a child with non-Hodgkin s lymphoma presenting as a focal unilateral renal mass with bony metastases.

Identification of the diastereomers of pentobarbital N-glucosides excreted in human urine.

A study was undertaken to determine if humans excreted pentobarbital N-glucosides as urinary metabolites following oral administration of pentobarbital. (1'RS,5RS)-1-(beta-D-Glucopyranosyl)pentobarbital ((1'RS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (1'RS,5R)-PTBG and (1'RS,5S)-PTBG were separated and found to be identical to synthetic standards when compared using HPLC retention times coupled with UV (with and without post-column ionization) and mass spectrometry (HPLC/MS). A HPLC method was developed for detecting and quantifying (1'RS,5R)-PTBG, (1'RS,5S)-PTBG and pentobarbital in urine. Following a single oral dose of sodium pentobarbital to male subjects (n = 6), 1.6-6.2% of the pentobarbital dose was excreted as (1'RS,5S)-PTBG over 60 hours. (1'RS,5R)-PTBG was also detected in one subject and accounted for 0.3% of the pentobarbital dose. Using a modified HPLC system, the four pentobarbital N-glucosides were resolved and analysis of a partially purified pentobarbital N-glucoside extract from one subject indicated that only (1'R,5R)-PTBG and (1'S,5S)-PTBG could be detected as urinary excretion products. These results indicate that the side chain chirality of pentobarbital may influence the observed enantioselectivity for the formation and/or urinary excretion of the pentobarbital N-glucosides.

Easily missed injuries around the knee.

Most fractures around the knee are easily detected on high-quality radiographs. However, some fractures and musculotendinous and ligamentous injuries have subtle findings and may be difficult to detect even with optimal images; these injuries include tibial plateau fractures, Segond fractures, stress fractures, fibular head fractures and dislocations, injuries to the patella and extensor mechanism, and Salter type fractures. Clinically suspected tibial plateau fractures unseen on standard views may be seen on tangential or tunnel projections. Segond fractures usually have a characteristic appearance on anteroposterior radiographs but occasionally are seen only on tunnel views. Stress fractures of the proximal tibia may be accompanied by a vague band of increased sclerosis or endosteal callus on either side of the epiphyseal scar. Correct diagnosis of fibular head dislocations requires clinical suspicion, since these injuries often are not recognized on initial radiographs. Careful evaluation of the congruity of the tibiofibular joint on the lateral projection is the key to diagnosis. Vertical patellar fractures are often nondisplaced and are best evaluated with sunrise or Merchant views; avulsion fractures from the proximal or distal poles, with lateral views; and osteochondral fractures, with sunrise or internal oblique views. Salter I injuries can be visualized on oblique and anteroposterior views obtained with stress applied to the knee. Some occult Salter I fractures are diagnosed on follow-up radiographs, which show periosteal reaction. Imaging modalities other than radiography are rarely needed to diagnose fractures but are useful for evaluating the extent of displacement or confirming soft-tissue injuries.

Ab initio association with beta 2-microglobulin during biosynthesis of the H-2Ld class I major histocompatibility complex heavy chain promotes proper disulfide bond formation and stable peptide binding.

In vitro translation studies indicate that the beta 2-microglobulin (beta 2-m) light chain influences the formation of intrachain disulfide bonds in class I major histocompatibility complex (MHC) molecules during their biosynthesis. We now have examined the influence of beta 2-m on class I MHC intrachain disulfide bond formation in vivo. Using beta 2-m+ and beta 2-m- derivatives of a cell line transfected with the mouse H-2Ld gene, we show that all of the H-2Ld molecules from beta 2-m+ cells have both the alpha 2 and alpha 3 intrachain disulfide bonds, whereas about 50% of the H-2Ld molecules from beta 2-m- cells have only one of these bonds. All of the free H-2Ld heavy chains from beta 2-m+ cells can undergo a peptide-induced conformational change and can bind exogenous peptide and beta 2-m stably in vitro. Only those H-2Ld molecules from beta 2-m- cells, which have both intrachain disulfide bonds, undergo a peptide- and beta 2-m-induced conformational change in vitro. These H-2Ld molecules do not bind beta 2-m and peptide stably in vitro. From these results emerges a greater understanding of the role of beta 2-m at the time of class I MHC molecule synthesis: beta 2-m promotes intrachain disulfide bond formation in the class I MHC molecule and additionally affects class I MHC structure to render it competent to form stable trimolecular complexes with peptide and beta 2-m.

Class I histocompatibility molecule association with phosphorylated calnexin. Implications for rates of intracellular transport.

Recent studies have shown that the endoplasmic reticulum (ER)-resident protein, calnexin, associates with class I major histocompatibility complex (MHC) molecules early in their biosynthesis. It has been suggested that calnexin participates in the assembly of class I MHC molecules or in the retention within the ER of unassembled class I molecules. We have examined the role of phosphorylation of calnexin in its association with mouse class I MHC molecules. We show that phosphocalnexin associates with H-2Ld and H-2Db molecules but not with H-2Kb and H-2Dd molecules, although calnexin-H-2Kb association can be demonstrated. These observations are interesting in light of the fact that H-2Kb and H-2Dd molecules are transported out of the ER more rapidly than are H-2Ld and H-2Db molecules. H-2Ld and H-2Db molecules differ in amino acid sequence only in their membrane-distal alpha 1 and alpha 2 domains. Nevertheless, the affinity of phosphocalnexin for H-2Ld is greater than its affinity for H-2Db. Furthermore, H-2Db becomes endoglycosidase H-resistant more slowly in cells in which it associates with phosphocalnexin than in cells in which it does not. Ca2+ ionophore A23187 prevents association of phosphocalnexin with H-2Ld molecules in vivo but does not cause the disruption of phosphocalnexin-H-2Ld complexes after they have formed. A23187 does not prevent assembly of H-2Ld-beta 2-microglobulin (beta 2-m) heterodimers. Furthermore, phosphocalnexin is found associated with H-2Ld molecules regardless of their state of assembly with beta 2-m and antigenic peptide. These results suggest that phosphocalnexin association with class I MHC molecules does not play a role in assembly of the class I MHC-beta 2-m-peptide complex nor in preventing release of unassembled class I molecules from the ER but may otherwise influence their rate of transport through the ER.

The cytoplasmic domain of the H-2Ld class I major histocompatibility complex molecule is differentially accessible to immunological and biochemical probes during transport to the cell surface.

An antiserum was generated against a synthetic peptide corresponding to a portion of the cytoplasmic domains of the H-2Ld and H-2Db class I major histocompatibility complex molecules of the mouse. This antibody preparation, R4, binds exclusively to endoglycosidase H-resistant H-2Ld/Db molecules which are not associated with beta 2-microglobulin. Interestingly, acquisition of resistance to endoglycosidase H precedes acquisition of R4 reactivity by 30 min. R4-reactive H-2Ld and H-2Db molecules occur on the cell surface and are phosphorylated in vivo. Other studies show that the tyrosine in the cytoplasmic domain is accessible to radioiodination on only a subset of H-2Ld molecules, and that the two-dimensional electrophoretic profiles of phosphorylated H-2L/Db molecules, of R4-reactive molecules, and of H-2Ld molecules radiolabeled on this cytoplasmic domain tyrosine are virtually identical. R4-reactive H-2Ld molecules do not undergo the peptide- and beta 2-microglobulin-induced conformational changes characteristic of free class I major histocompatibility complex heavy chains. The accessibility of the H-2Ld cytoplasmic domain to R4 and to radioiodination late in biosynthesis and its biological significance are discussed.

In vivo dimeric association of class I MHC heavy chains. Possible relationship to class I MHC heavy chain-beta 2-microglobulin dissociation.

Class I MHC molecules have been thought to occur in vivo both as class I MHC heavy chain-beta 2-m heterodimers, which are or are not associated with antigenic peptide, and as free class I MHC heavy chains. Class I MHC molecules are now found also to occur in another type of structure: a heavy chain-heavy chain dimer. Biochemical studies show that heavy chain dimers are disulfide-linked via a conserved cytoplasmic domain cysteine. H-2Ld, H-2Db, and H-2Dd class I dimers fail to react with certain alpha 1 and alpha 2 domain-specific antibodies. Furthermore, although beta 2-m-specific antibodies coprecipitate class I MHC heavy chains, they do not coprecipitate class I MHC heavy chain dimers. Pulse-chase studies show that heavy chain dimer formation occurs at different points in the biosynthesis of class I MHC molecules in beta 2-m+ and beta 2-m- cells: in beta 2-m+ cells, heavy chain dimers form after the class I molecules have traversed the medial Golgi cisternae, whereas in beta 2-m- cells they form immediately. Culturing of beta 2-m+ cells with exogenous beta 2-m prevents the formation of H-2Ld/Db heavy chain dimers. We conclude that dimer formation occurs as a consequence of loss or unavailability of beta 2-m. Class I MHC heavy chain dimerization may provide a mechanism for removal of immunologically dysfunctional molecules.

Effects of proteolytic and thermal action on the activity of lactate dehydrogenase isozymes LD-1 and LD-5.

L-Lactate dehydrogenase (LD) catalyzes the interconversion of pyruvate and lactate. Using a spectrophotometric assay to determine LD activity, incubation of rabbit, porcine, and bovine LD-1 and LD-5 isozymes with the protease subtilisin (Carlsberg) gave first-order degradation kinetics. Degradation half-lives were significantly lower for the LD-5 isozymes from the three species when incubated with subtilisin at temperatures from 4 degrees C to 25 degrees C. The energy involved in the degradation process, however, was not different. The activation energy for the conversion of pyruvate to lactate by LD-1 at pH 7.4 was significantly higher than that for LD-5 for all three species examined (P less than 0.005). Thermocalorimetry showed that the LD-1 isozymes have both a higher mean temperature of denaturation and a higher heat uptake during the denaturation process than corresponding LD-5 forms. The results suggest that the LD-5 isozymes in the species studied are more metabolically efficient, whereas the LD-1 forms have greater structural stability.

A double-labeling method for measuring induction of protein phosphorylation.

Protein phosphorylation is widely believed to play a regulatory role in signal transduction, mitosis, cell proliferation, cell motility, cell shape, gene regulation, and many other cellular processes. Thus, the quantitation of phosphorylation of specific cellular proteins may provide insight into the mechanisms by which phosphorylation is employed in regulation. Moreover, identification of phosphorylation substrates of various cellular kinases provides an important first step in determining their role in cellular regulation. However, accurate measurement of the differential phosphorylation of cellular proteins under different physiological conditions is often difficult to achieve. To address this problem, we have developed an in vivo double-labeling protocol (utilizing [3H]-, [14C]-, or [35S]-radiolabeled amino acids and [32P]-orthophosphate) that allows the quantitation of the amount of specific phosphorylation of a given protein from densitometric analysis of autoradiograms of polyacrylamide gels. This double-labeling strategy provides a means of quantitating the phosphorylation of individual biosynthetically labeled proteins. This method can be used in the analysis of immunoprecipitated proteins, proteins from subcellular fractions, such as nuclei or selected membrane fractions, or even total cellular proteins displayed on two-dimensional gels.

Endocytosis of the class I major histocompatibility antigen via a phorbol myristate acetate-inducible pathway is a cell-specific phenomenon and requires the cytoplasmic domain.

Class I major histocompatibility (MHC) antigens are expressed by virtually all mammalian cells, yet their levels of expression and behavior on the cell surface vary in a cell-specific fashion. A panel of lymphoid (both B and T) and nonlymphoid cell lines was used to study the kinetics of internalization of the H-2Ld class I MHC in different cell types. These studies revealed that endocytosis of H-2Ld occurs by both constitutive and PMA-regulated pathways in lymphoid cells, but only by a PMA-refractory pathway in the nonlymphoid cells tested. Transfectant derivatives of the T lymphoma, EL4, which express wild-type or mutant H-2Ld class I MHC antigens, were used to investigate the requirement for the cytoplasmic domain of the class I MHC antigen for its endocytosis in T lymphocytes. These studies showed that modification or deletion of the cytoplasmic domain of H-2Ld abrogates endocytosis via a PMA-regulated pathway. The role of cytoplasmic domain phosphorylation in PMA-inducible endocytosis was examined. The wild-type H-2Ld antigen is phosphorylated in all cell types examined, and this phosphorylation is up-regulated by PMA treatment. In contrast, cytoplasmic domain mutants of H-2Ld fail to be phosphorylated in vivo, in the presence or absence of PMA. The universality of PMA-inducible hyperphosphorylation of the class I MHC antigen among diverse cell types leads us to conclude that phosphorylation of the cytoplasmic domain, while perhaps necessary, is not sufficient for triggering endocytosis via a PMA-inducible pathway. Furthermore, the results with the cytoplasmic domain mutants of H-2Ld suggest that a structural conformation of the class I MHC cytoplasmic domain is required for endocytosis via this route.