Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis.

The human serum protein transthyretin (TTR) is highly fibrillogenic in vitro and is the fibril precursor in both autosomal dominant (familial amyloidotic polyneuropathy [FAP] and familial amyloidotic cardiomyopathy [FAC]) and sporadic (senile systemic amyloidosis [SSA]) forms of human cardiac amyloidosis. We have produced mouse strains transgenic for either wild-type or mutant (TTRLeu55Pro) human TTR genes. Eighty-four percent of C57BI/6xDBA/2 mice older than 18 months, transgenic for the wild-type human TTR gene, develop TTR deposits that occur primarily in heart and kidney. In most of the animals, the deposits are nonfibrillar and non-Congophilic, but 20% of animals older than 18 months that bear the transgene have human TTR cardiac amyloid deposits identical to the lesions seen in SSA. Amino terminal amino acid sequence analysis and mass spectrometry of the major component extracted from amyloid and nonamyloid deposits revealed that both were intact human TTR monomers with no evidence of proteolysis or codeposition of murine TTR. This is the first instance in which the proteins from amyloid and nonfibrillar deposits in the same or syngeneic animals have been shown to be identical by sequence analysis. It is also the first time in any form of amyloidosis that nonfibrillar deposits have been shown to systematically occur temporally before the appearance of fibrils derived from the same precursor in the same tissues. These findings suggest, but do not prove, that the nonamyloid deposits represent a precursor of the fibril. The differences in the ultrastructure and binding properties of the deposits, despite the identical sizes and amino terminal amino acid sequences of the TTR and the dissociation of deposition and fibril formation, provide evidence that in vivo factors, perhaps associated with aging, impact on both systemic precursor deposition and amyloid fibril formation.

Gamma heavy chain disease in man: independent structural abnormalities and reduced transcription of a functionally rearranged lambda L-chain gene result in the absence of L-chains.

Human Ig heavy chain diseases of the alpha and gamma classes are characterized by the absence of light chain production as well as the disease-defining abnormalities in the heavy chain protein. Prior studies have suggested concomitant structural defects in productively rearranged L-chain genes as the reason for the absent L-chain proteins. We have found that the single rearranged lambda L-chain gene in the OMM heavy chain disease (HCD) cell line has a mutation in the splice donor site at the 3' end of the J exon, resulting in direct splicing of the 3' end of the leader to the acceptor site of the constant region. The cells contain an mRNA consisting of the leader-coding region joined directly to the constant region. The V-region exon is skipped and the shortened mRNA is translated into a truncated protein containing no V-region amino acids. We have also noted that, in contrast to most normal and neoplastic Ig-producing cells, the OMM cells produce an excess of heavy to light chain mRNA as well as protein. The excess is independent of the structural gene abnormality and is due to a low level of L-chain transcription, which can be increased by fusing the HCD cell to the murine myeloma cell line NS-1 or transfecting the defective OMM L-chain gene into a murine plasma cell. The latter data suggest that the OMM cells either lack a transcription factor present in mature plasma cells or have a functional repressor of L-chain transcription.

Prostate cancer: local staging with endorectal magnetic resonance imaging.

BACKGROUND: Prostate cancer has received increasing attention during the past decades. Staging of tumors before treatment is imperative for planning appropriate therapy. The purpose of this study is to assess the role of endorectal magnetic resonance imaging (MRI) in local staging of prostate cancer. METHODS: Endorectal MRI was performed in 31 patients with histologically-proven prostate cancer. MRI was done three to 100 days (mean, 32.1 days) after either transrectal ultrasonography (TRUS) with biopsy or transurethral resection of the prostate (TURP). Radical prostatectomies were performed within two weeks after MRI. The diagnostic accuracy of endorectal MRI for local tumor staging, specifically for extracapsular extension (ECE) and seminal vesicle invasion (SVI), was evaluated by correlating MRI results with histopathologic findings of whole-mount specimens. RESULTS: The accuracy of endorectal MRI for the detection of tumor presence and estimation of tumor volume was 48%. Sensitivity, specificity and positive predictive value for evaluation of ECE were 88%, 69% and 80%, respectively, and for SVI, were 66%, 84% and 50%, respectively. The overall accuracy of MRI in local tumor staging (using the TMN system) was 61%. Accuracy in differentiating localized from invasive cancer was 84%. CONCLUSION: Endorectal MRI is not accurate enough to detect tumor presence or estimate tumor volume. Diagnostic accuracy for local tumor staging is unsatisfactory. However, endorectal MRI is highly accurate in differentiating localized (stage B) from invasive (stage C) cancer.

Pituitary apoplexy diagnosed by magnetic resonance imaging: a case report.

Pituitary apoplexy is a neuroendocrine emergency produced by hemorrhage or infarction of pituitary tumors, and its accurate diagnosis has relied mainly on clinical manifestations and computed tomography (CT) scan of sella in the past. A case is reported of pituitary apoplexy which was demonstrated on magnetic resonance imaging (MRI). The 72-year-old male patient was admitted to the Nephrology Section of VGH-Taipei with symptoms of general malaise, poor appetite for four months and apparent hyponatremia (Na 102 mEq/L) on laboratory findings. Under the impression of syndrome of inappropriate secretion of antidiuretic hormone (SIADH), fluid restriction and infusion of isotonic saline were prescribed, but in vain. The patient was transferred to the Endocrinology Section because of lowered serum cortisol, T3, T4, and hs-TSH levels. Dynamic pituitary function tests confirmed the diagnosis of panhypopituitarism. Contrast enhanced CT scan of sella revealed displacement of the pituitary stalk to the right side. T1-weighted MRI showed persistent high intensity in left sella and T2-weighted MRI showed persisting isointense to white matter. The hyponatremia and clinical symptoms resolved within a few days after replacement therapy with glucocorticoid and thyroid hormone. It was concluded that MRI is more sensitive than CT scan for detecting subacute or chronic pituitary apoplexy.

Percutaneous transluminal angioplasty of neck vessels.

BACKGROUND: Although surgical endarterectomy or bypass is regarded as the treatment-of-choice for extracranial cerebral vascular stenosis, percutaneous transluminal angioplasty (PTA) has its role as an alternative procedure for symptomatic patients refractory to medical therapy and/or for whom surgical treatment is contraindicated. METHODS: Occlusive blood vessels were first identified by neurologic, ultrasonic and angiographic studies. Patients were premedicated with heparin and corticosteroid. From a transfemoral approach, double lumen balloon dilatation catheter was guided across the area of stenosis, and inflated two or three times to produce dilatation. Following PTA, patients received anticoagulants and corticosteroid for a period, and were followed by clinical examination, Doppler blood-flow and angiographic studies. RESULTS: Seventeen patients undergoing 26 procedures were included in this study. The lesions involved subclavian artery in nine cases, common carotid artery in seven, internal carotid artery in five and proximal vertebral artery in five. Digital subtraction angiography (DSA) was done immediately after PTA and showed improvement of stenotic segment (residual stenosis < 30%) in 22 vessels. Restenosis occurred in two cases of subclavian artery PTA six months later. There were three complications, including TIA in two cases and hematoma at the puncture site in one case. Clinically, symptomatic improvement was obtained in 13 cases. CONCLUSIONS: In patients with significant atherosclerotic stenosis of neck vessels, PTA is a beneficial procedure for improving blood flow to brain circulation and for alleviating symptoms.

A Thy-1 negative lymphoma cell variant defective in the formation of glycosyl-phosphatidylinositol membrane protein anchors.

Thy-1 is a glycoprotein present on the membrane of murine cells of the T-lineage. The mature Thy-1 is anchored to the membrane via a glycolipid, phosphatidylinositide. In order to study the regulation of the synthesis and membrane insertion of this protein, the biochemical properties of a Thy-1.2 negative variant T-lymphoma cell (RL male 1.4) were studied. It contains intracellular Thy-1 protein but fails to express it on the cell surface. While the wild type and the mutant show similar labelling of the intracellular Thy-1 glycoprotein with amino acids, no ethanolamine is incorporated into the Thy-1 molecule of RL male 1.4. A plasmid, pT1, containing the normal Thy-1.2 gene and bacterial gpt gene was transfected into RL male 1.4 and into the murine plasmacytoma cell, J558L. A transfected plasmacytoma, T1J2, synthesized a normal sized Thy-1 protein and displayed the antigen on the membrane. In contrast, the mycophenolic acid resistant RL male 1.4 transfectants did not display Thy-1.2 on the cell surface, despite the presence of substantial amounts of Thy-1 intracellularly. Two other antigens known to be anchored in the membrane by phospholipid, Ly-6e and Qa-2, were also examined in RL male 1.4. RL male 1.4 did not express Ly-6e after alpha interferon induction. In addition, the expression of Qa-2 antigen was greatly diminished in RL male 1.4 in comparison to RL male 1.3. Thus, the defect in RL male 1.4 is not restricted to Thy-1.2, but includes other similarly anchored glycoproteins as well. This implies that the addition of phospholipid to core proteins is similar, if not identical, for all these molecules and that the RL male 1.4 cell lacks the capacity to from the lipid glycoprotein linkage required for the expression of these proteins on the cell surface.

Fibronectin from chicken embryo fibroblasts contains covalently bound phosphate.

Fibronectin isolated from cultures of chicken embryo fibroblasts (CEF) contains phosphorus linked to serine and threonine by monoester bonds. Normal and Rous sarcoma virus (RSV)-transformed cells were incubated with [32P]orthophosphate, and fibronectin was isolated from the cell surfaces and conditioned media. 32P was stably associated with fibronectin during immunoprecipitation, SDS-polyacrylamide gel electrophoresis, phospholipid solvent extraction, and hot acid but not alkaline treatment. After a limited acid hydrolysis of fibronectin, both phosphoserine and phosphothreonine were found. The specific radioactivity of the 32P-labeled fibronectin from the conditioned medium of normal CEF was higher than that from the cultures of transformed CEF.

Insulin effect on the cell cycle: analysis of the kinetics of growth parameters in confluent chick cells.

Several techniques, including flow microfluorometry, were utilized to study the effect of insulin on the growth of cultured cells. It was demonstrated that chick fibroblasts can be stimulated to synthesize DNA and undergo mitosis after insulin addition. The kinetics of the cell movement through the cell cycle as well as the length of the cycle itself, however, were distinctly different in insulin- and serum-treated cultures. The insulin-treated cells had a shorter G1, an extended S, and a much extended G2 residence time compared to cells treated with serum. A model of growth regulation which includes both primary cultures and cell lines is proposed.

Interaction of beef liver lipase with mixed micelles of tripalmitin and Triton X-100.

When concentrated dispersions of tripalmitin in Triton X-100 are added to reaction mixtures containing soluble beef liver lipase, the rate of hydrolysis of tripalmitin increases with incubation time. When the diluted substrate is aged at 37 degrees C for 3 hr before the addition of enzyme, the rate of hydrolysis is greater than the rate with freshly diluted dispersions and is constant for at least 2 hr. The reciprocal of the rate of hydrolysis is a complex function of the reciprocal of the substrate concentration when measured with freshly diluted substrate dispersions. A linear relationship between these reciprocals is obtained when measured with aged preparations of substrate. The rate and extent of increase of the velocity of hydrolysis of the aged substrate in relation to the velocity of hydrolysis of freshly diluted substrate are directly proportional to the substrate concentration and inversely proportional to the Triton X-100 concentration. The apparent V(max) of beef liver lipase for tripalmitin in diluted and aged dispersions is independent of the Triton X-100 concentration, while the apparent K(m) is inversely proportional to the Triton X-100 concentration. The apparent K(m) for tripalmitin complexes at zero Triton X-100 concentration was judged to be 7.5 x 10(-5) m. The molecular size of dispersion complexes does not change significantly as dispersions are aged. The spherical diameter of the complexes assessed by gel filtration techniques is in the order of 100 A.