Oligomeric structure of type I and type II transforming growth factor beta receptors: homodimers form in the ER and persist at the plasma membrane.

Transforming growth factor beta (TGF-beta) signaling involves interactions of at least two different receptors, types I (TbetaRI) and II (TbetaRII), which form ligand-mediated heteromeric complexes. Although we have shown in the past that TbetaRII in the absence of ligand is a homodimer on the cell surface, TbetaRI has not been similarly investigated, and the site of complex formation is not known for either receptor. Several studies have indicated that homomeric interactions are involved in TGF-beta signaling and regulation, emphasizing the importance of a detailed understanding of the homooligomerization of TbetaRI or TbetaRII. Here we have combined complementary approaches to study these homomeric interactions in both naturally expressing cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both TbetaRI and TbetaRII form homodimer-sized complexes in the endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors carrying different epitope tags, we have demonstrated ligand-independent homodimers of TbetaRI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains are not sufficient to mediate homodimerization, although TGF-beta1 is able to promote dimerization of the type II receptor ectodomain. These findings may have important implications for the regulation of TGF-beta signaling.

Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction.

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.

Cloning and chromosomal localization of a human kidney cDNA involved in cystine, dibasic, and neutral amino acid transport.

We have recently cloned, sequenced, and characterized a rat kidney cDNA (D2) that stimulates cystine as well as dibasic and neutral amino acid transport. In order to evaluate the role of this protein in human inherited diseases such as cystinuria, we have isolated a human D2 clone (D2H) by low stringency screening of a human kidney cDNA library using the radiolabeled D2 insert as a probe. The D2H cDNA is 2284 nucleotides long and encodes a 663 amino acid protein that is 80% identical to the rat D2 amino acid sequence and 86% to that of the rabbit homologue rBAT. Microinjection of in vitro transcribed D2H cRNA into Xenopus oocytes induced uptake of cystine as well as dibasic and neutral amino acids in a pattern similar to that of rat D2 and rabbit rBAT. Both neutral and dibasic amino acids inhibited the D2H-induced uptake of cystine. Northern blot analysis demonstrated that D2H, like D2 and rBAT, is expressed strongly in the kidney and intestine. Southern blot analysis of genomic DNA from a panel of mouse-human somatic cell hybrids showed that the human gene for D2H resides on chromosome 2.

Increased ADAMTS-13 proteolytic activity in rat hepatic stellate cells upon activation in vitro and in vivo.

INTRODUCTION: ADAMTS-13 is a member of A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, primarily synthesized in hepatic stellate cells (HSCs), one of the major cell types transdifferentiating into myofibroblasts during liver fibrosis. However, the association between ADAMTS-13 expression and HSC activation or liver fibrosis is not known. METHODS: In this study, we determined the ADAMTS-13 mRNA, protein, and activity in isolated primary HSCs upon activation on a plastic dish and in liver after administration of carbon tetrachloride (CCl(4)) in rats. RESULTS: We showed that ADAMTS-13 antigen and proteolytic activity in the activated rat HSCs were dramatically increased, whereas ADAMTS-13 mRNA in these cells was only minimally altered. Similarly, the ADAMTS-13 antigen and proteolytic activity in rat liver after CCl(4) injury were also significantly increased, whereas the ADAMTS-13 mRNAs in these liver tissues were only slightly increased compared with normal. Surprisingly, despite the dramatic up-regulation of ADAMTS-13 protein synthesis in the activated HSCs after CCl(4) administration, the plasma levels of ADAMTS-13 protease in rats did not increase concordantly. CONCLUSION: We conclude that the up-regulation of ADAMTS-13 protein expression in rat HSCs during activation in vitro and in vivo suggests the possibility of ADAMTS-13 proteolysis, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries. The data also suggest the minimal contribution of the activated HSCs in regulation of plasma levels of ADAMTS-13 protease.

Analysis of the gamma delta res site. Sites required for site-specific recombination and gene expression.

The gamma delta resolvase, product of the transposon's tnpR gene, mediates a site-specific recombination between two copies of gamma delta directly repeated on the same replicon. The site at which the recombination occurs, res, lies between the tnpA and tnpR genes. Within this same region lie the promoters for expression of tnpA and tnpR and the operator sites through which resolvase regulates the transcription. In order to determine the extent of the res site we have constructed in vitro a series of deletions that terminate within the tnpA-tnpR intergenic region, and have analyzed their effect on site-specific recombination. Our results indicate that a fully functional res site is about 115 base-pairs (bp) and runs from a position 15 bp to the left (tnpA-proximal) side of the crossover point to 100 bp to the right. This segment corresponds precisely to the region defined by the three resolvase binding sites that we have demonstrated previously. Alterations of the nucleotide sequence around the crossover point indicate the importance of all or part of the central palindrome 5' T-T-A-T-A-A within which the breakage--reunion reaction takes place. Taken together, our results strengthen our earlier conclusion that resolvase recognizes the 9 bp segment 5' T-G-T-C-Y-N-N-T-A that occurs (in slightly degenerate form) in each half of the three binding sites. Using the deletions we have confirmed that the tnpA promoter spans the crossover site and have shown that the major tnpR promoter in vivo coincides with resolvase binding site II, although a second promoter for tnpR transcription lies across site I.

Comparing filtered backprojection and ordered-subsets expectation maximization for small-lesion detection and localization in 67Ga SPECT.

UNLABELLED: Iterative reconstruction of SPECT images has recently become clinically available as an alternative to filtered backprojection (FBP). However, there is conflicting evidence on whether iterative reconstruction, such as with the ordered-subsets expectation maximization (OSEM) algorithm, improves diagnostic performance over FBP. The study objective was to determine if the detection and localization of small lesions in simulated thoracic gallium SPECT images are better with OSEM reconstruction than with FBP, both with and without attenuation correction (AC). METHODS: Images were simulated using an analytic projector acting on the mathematic cardiac torso computer phantom. Perfect scatter rejection was assumed. Lesion detection accuracy was assessed using localization receiver operating characteristic methodology. The images were read by 5 nuclear medicine physicians. For each reconstruction strategy and for each observer, data were collected in 2 viewing sessions of 100 images. Two-way ANOVA and, when indicated, the Scheffé multiple comparisons test were applied to check for significant differences. RESULTS: Little difference in the accuracy of detection or localization was seen between FBP with and without AC. OSEM with AC extended the contrast range for accurate lesion detection and localization over that of the other methods investigated. Without AC, no significant difference between OSEM and FBP reconstruction was detected. CONCLUSION: OSEM with AC may improve the detection and localization of thoracic gallium-labeled lesions over FBP reconstruction.

Spine pain in children.

Spine pain in children as an isolated symptom is infrequently seen in clinical practice. However, as opposed to spine pain in adults, it is accompanied by a relatively higher frequency of pathology. Diagnostic imaging is a necessary follow up to the physical examination and laboratory studies. Standard radiography is usually the initial imaging study. Frequently, however, additional imaging with bone scintigraphy, computed tomography, or magnetic resonance imaging is necessary to add sensitivity and specificity to the clinical presentation. We describe the disease entities, pathophysiology, and imaging characteristics of the varied pathologies responsible for spine pain in children.

The child with acute leg pain.

Diagnostic imaging is an essential component in the evaluation of a child with acute lower extremity pain or limp. Most, but not all, of the etiologies are traumatic. Frequently, the history and clinical examination are not specific. Radionuclide bone imaging is extremely rewarding in these circumstances. It represents the most useful examination used in evaluating the pathophysiology and full extent of the disorder. This article discusses the clinical presentation, pathophysiology, and characteristic imaging observations in the more common problems associated with hip and/or leg pain.

Cerebral toxoplasmosis in an immunocompromised host. A precise and rapid diagnosis by electron microscopy.

In immunocompromised patients with cerebral toxoplasmosis, the tachyzoite forms rather than cystic and bradyzoite forms of the protozoon are commonly seen. These tachyzoites are minute, scattered among cellular debris, sometimes lodged inside macrophages and neutrophils, and difficult to visualize by light microscopy, even with special stains. Immunodiagnostic tests may be falsely negative due to inability of the host to produce appropriate antibodies. Isolation of the organism is dangerous because Toxoplasma gondii is highly infective. In this situation, transmission electron microscopy (EM) may be a diagnostic tool of choice. It demonstrates the fine definitive features of the protozoon and can be expedited to give results in five hours. Further evaluation of EM for diagnosing possible toxoplasmosis in immunocompromised patients is indicated.

Wire stent for tracheomalacia in a five-year-old girl.

A wire stent was used successfully to treat life-threatening tracheomalacia in a 5-year-old girl. Wire stents placed bronchoscopically are nonobstructing and have the potential for balloon expansion to accommodate growth.

Effect of filtering on the detection and localization of small Ga-67 lesions in thoracic single photon emission computed tomography images.

Tumor detection can be significantly affected by filtering so determining an optimal filter is an important aspect of establishing a clinical reconstruction protocol. The purpose of this study was to identify the cut-off frequency of a Butterworth filter used in a filtered backprojection (FBP) reconstruction that maximized the detection and localization accuracy of 1 cm spherical lesions in Ga-67 citrate, thoracic SPECT images. Image quality was evaluated by means of a localization receiver operating characteristic (LROC) study using computer simulated images. Projection data were generated using the mathematical cardiac-torso digital phantom with a clinically realistic background source distribution. The images were reconstructed using FBP with multiplicative Chang attenuation correction and fifth-order Butterworth filtering. The cut-off frequencies considered were 0.25, 0.32, 0.47, and 0.79 cm(-1) for the case of three-dimensional (3D) post-filtering and 0.25, 0.32, and 0.47 cm(-1) for two-dimensional (2D) post-filtering. The images were read by three research scientists and one board certified nuclear medicine clinician. The area under the LROC curve and the localization accuracy for all test conditions were compared using Scheffé's multiple comparisons test. It was found that 3D post-filtering using filters with cut-off frequencies of 0.32 and 0.47 cm(-1) resulted in the highest lesion detectability and localization accuracy. These two test conditions did not differ significantly from each other but were significantly better (p<0.05) than all of the 2D, and the 3D 0.79 cm(-1) cut-off frequency cases.

LROC analysis of detector-response compensation in SPECT.

Localization ROC (LROC) observer studies examined whether detector response compensation (DRC) in ordered-subset, expectation-maximization (OSEM) reconstructions helps in the detection and localization of hot tumors. Simulated gallium (Ga-67) images of the thoracic region were used in the study. The projection data modeled the acquisition of attenuated 93- and 185-keV photons with a medium-energy parallel-hole collimator, but scatter was not modeled. Images were reconstructed with five strategies: 1) OSEM with no DRC; 2) OSEM preceded by restoration filtering; 3) OSEM with iterative DRC; 4) OSEM with an ideal DRC; and 5) filtered backprojection (FBP) with no DRC. All strategies included attenuation correction. There were four LROC studies conducted. In a study using a single tumor activity, the ideal DRC offered the best performance, followed by iterative DRC, restoration filtering, OSEM with no DRC, and FBP. Statistical significance at the 5% level was found between all pairs of strategies except for restoration filtering and OSEM with no DRC. A similar ranking was found for a more realistic study using multiple tumor activities. Additional studies considered the effects of OSEM iteration number and tumor activity on the detection improvement that iterative DRC offered with respect to OSEM with no DRC.

Hemimegalencephaly: clinical, EEG, neuroimaging, and IMP-SPECT correlation.

Iofetamine-single photon emission computed tomography (IMP-SPECT) was performed on 2 girls (5 1/2 and 6 years of age) with histories of intractable seizures, developmental delay, and unilateral hemiparesis secondary to hemimegalencephaly. Electroencephalography (EEG) revealed frequent focal discharges in 1 patient, while a nearly continuous burst suppression pattern over the malformed hemisphere was recorded in the other. IMP-SPECT demonstrated a good correlation with neuroimaging studies. In spite of the different EEG patterns, which had been proposed to predict contrasting clinical outcomes, both IMP-SPECT scans disclosed a similar decrease in tracer uptake in the malformed hemisphere. These results are consistent with the pattern of decreased tracer uptake found in other interictal studies of focal seizures without cerebral malformations. In view of recent recommendations for hemispherectomy in these patients, we suggest that the IMP-SPECT scan be used to compliment EEG as a method to define the extent of abnormality which may be more relevant to long-term prognosis than EEG alone.

Extraosseous bone-tracer uptake. Plasma cell granuloma.

A case of a large plasma-cell granuloma of the left upper lobe in a 5-year-old girl is presented. Uptake of Tc-99m MDP by the soft tissue mass was demonstrated with bone imaging. Plasma cell granuloma should be included in the gamut of extraosseous uptake of bone-seeking radionuclide in the pediatric chest.

Comparative imaging. Disk calcification in childhood.

Intervertebral disk calcification in childhood probably occurs secondary to disk inflammation or trauma. Images obtained in the evaluation of a six-year-old boy with cervical disk calcification demonstrate the typical findings with radiography, bone scintigraphy, and computed tomography.

Suprarenal abscess in the neonate. Technetium-99m glucoheptonate imaging.

Although suprarenal abscess in a newborn is rare, a prompt diagnosis is essential for proper patient management. The findings obtained with Tc-99m glucoheptonate renal imaging in a newborn with a right adrenal abscess are reported. A radionuclide renal imaging sequence over a 15-hour period demonstrated a "rim sign" which can be used to suggest the diagnosis. Radionuclide and ultrasound imaging of neonatal adrenal masses is discussed.

Myocardial imaging. Coxsackie myocarditis.

A 3-week-old male neonate with heart failure associated with Coxsackie virus infection was imaged with Tc-99m PYP and TI-201. The abnormal imaging pattern suggested myocardial infarction. Autopsy findings indicated that the cause was myocardial necrosis secondary to an acute inflammatory process. Causes of abnormal myocardial uptake of Tc-99m PYP in pediatrics include infarction, myocarditis, cardiomyopathy, bacterial endocarditis, and trauma. Myocardial imaging cannot provide a specific cause diagnosis. Causes of myocardial infarction in pediatrics are listed in Table 1.

Lymphoscintigraphy. Lower extremity lymphangioma.

Congenital lymphangioma is a relatively common cause of childhood soft tissue masses of the extremities. Radionuclide pedal lymphangiography is a simple and rapid technique for the diagnosis of lymphangiomas of the lower extremities. A case of congenital lymphangioma of the thigh in an eight-year-old child is illustrated.

Scintigraphic patterns in osteoid osteoma and spondylolysis.

A retrospective review was performed of radionuclide bone scans and radiographs of 54 children with diagnoses of spondylolysis or osteoid osteoma/osteoblastoma. The findings indicate that skeletal scintigraphy shows imaging patterns that can be used to help distinguish these two conditions. Most cases of spondylolysis and all cases of osteoid osteoma or osteoblastoma demonstrated focally increased activity on the delayed skeletal scintigrams. Evaluation of the bone scan images obtained during the immediate postinjection period revealed intense activity with osteoid osteoma and osteoblastoma, but minimal or no abnormal activity in cases of spondylolysis. In addition, spondylolysis was the only condition that produced bilateral abnormalities in the pars interarticularis region in these children.

Managing miscarriage. The need for more than medical mechanics.

A compassionate physician recognizes the emotional as well as the physical needs of a woman who miscarries. The physical trauma is often short-lived, but the emotional aftermath may be prolonged. Dr Wells reviews the differential diagnosis and management of miscarriage and discusses the valuable role that a physician may take in alleviating the patient's feelings of guilt, grief, and frustration.