Estimation of lymph node metastasis by size in patients with intrathoracic oesophageal cancer.

BACKGROUND: The preoperative evaluation of lymphatic metastasis in patients with oesophageal cancer is of vital importance in determining a therapeutic strategy. The aim of this study was to establish criteria for the preoperative diagnosis of lymph node metastases based on the size and shape of nodes. METHODS: Some 123 patients with intrathoracic oesophageal cancer were studied and 6822 nodes were obtained by extended lymphadenectomy. The nodes were classified anatomically and their size was measured by the operating surgeon during or immediately after operation. All were examined histologically and criteria for the diagnosis of metastasis were evaluated. RESULTS: The size of the lymph nodes varied by anatomical site. Nodes were smallest in the neck and largest at the tracheal bifurcation. The cut-off value for the diagnosis of metastasis was 5 mm in the neck, 6 mm in the abdomen and 8 mm in the mediastinum, except for tracheal nodes. Lymph nodes 10 mm or larger tended to become spherical when involved by metastasis. CONCLUSION: The incidence of metastasis in each lymph node can be estimated by its size. Discounting nodes less than 10 mm can lead to an underestimation of the stage of oesophageal cancer during preoperative evaluation.

Peroxisomal and mitochondrial targeting of serine:pyruvate/alanine:glyoxylate aminotransferase in rat liver.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, SPTp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.

Induction by peroxisome proliferators and triiodothyronine of serine:pyruvate/alanine:glyoxylate aminotransferase of rat liver.

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.

In vitro association with peroxisomes and conformational change of peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase in rat and human livers.

To understand the targeting mechanisms of peroxisomal serine:pyruvate aminotransferase, in vitro import experiments were carried out using this 43 kDa peroxisomal enzyme, which was synthesized in a coupled transcription/translation system. Being different from other peroxisomal enzymes, such as acyl-CoA oxidase and urate oxidase used in previous in vitro import experiment, the soluble form of peroxisomal serine:pyruvate aminotransferase was fairly resistant to proteinase K digestion. However, the enzyme recovered in the peroxisomal fraction was proteinase K sensitive. This indicates that the association of peroxisomal serine:pyruvate aminotransferase with the peroxisomal membrane causes a marked conformational change in the structure of this protein.

Fidelity of translation initiation of mRNA for the precursor of rat mitochondrial serine:pyruvate/alanine:glyoxylate aminotransferase.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) of rat liver is localized in both mitochondria and peroxisomes. The rat SPT/AGT gene is single, but there are two species of mRNA which differ at their 5' termini due to transcription from two alternative initiation sites. The longer mRNA is translated from the first AUG codon and thereby directs synthesis of the 45 kDa precursor of mitochondrial SPT/AGT, which includes a mitochondria-targeting N-terminal signal sequence. Peroxisomal SPT/AGT is synthesized as a product of mature size (43 kDa) from the shorter mRNA, which starts 3' to the first AUG codon and thus is translated from a downstream AUG codon. In our previous immunocytochemical study, SPT/AGT was found to be localized only in peroxisomes, when a cDNA encoding 43 kDa SPT/AGT was expressed in COS cells. When a cDNA encoding the 45 kDa precursor was expressed, on the other hand, SPT/AGT was localized mostly in mitochondria, but a small number of peroxisomes were also positively stained [Yokota, S., Funai, T., and Ichiyama, A. (1991) Biomed. Res. 12, 53-59]. We show in this paper that 43 kDa SPT/AGT is also synthesized from the longer mRNA in an in vitro translation system through a leaky scanning mechanism. Although the first AUG initiator codon is in a suboptimal context, the amount of 43 kDa SPT/AGT synthesized from the longer mRNA was small, probably because a downstream stem-loop structure facilitates recognition of the first AUG initiator codon.

Urate oxidase is imported into peroxisomes recognizing the C-terminal SKL motif of proteins.

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Regulation by glucagon of serine: pyruvate/alanine: glyoxylate aminotransferase gene expression in cultured rat hepatocytes.

The effects of glucagon on serine: pyruvate/alanine: glyoxylate aminotransferase (SPT/AGT) gene expression were studied in primary cultured rat hepatocytes. When hepatocytes had been precultured for 16-18 h under serum- and hormone-free conditions, the addition of glucagon caused (after a lag period of about 2 h) a remarkable increase in the cellular level of SPT/AGT mRNA by 4 h in a time- and dose-dependent manner. The induced mRNA was that for mitochondrial SPT/AGT, as judged by ribonuclease protection analysis. A nuclear run-on assay revealed that activation of transcription is responsible for the increase in mitochondrial SPT/AGT mRNA and that the maximal rate of transcription occurs 1.5 h after glucagon addition. The effect of glucagon was mimicked by 8-bromo-cAMP and suppressed by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (protein kinase A), while both 12-O-tetradecanoylphorbol-13-acetate and A23187 were without effect in elevating the SPT/AGT mRNA level, suggesting that the cAMP/protein kinase A system is involved in the regulation of SPT/AGT gene expression. In hepatocytes precultured for 16-18 h under serum- and hormone-free conditions, the glucagon-induced transcription was severely inhibited by cycloheximide. When the preculture was for 2 h, on the other hand, the activation of transcription by glucagon was more rapid, and the inhibition by cycloheximide was less than that observed with cells precultured for 16-18 h, suggesting that a short-lived protein factor is involved in the hormonal regulation. The glucagon-induced expression of the SPT/AGT gene was also turned off by dexamethasone.

Regulation of glucokinase gene expression in cultured rat islet cells: the inhibitory effects of T3 and glucagon, and the stimulatory effect of glibenclamide.

Hormonal and non-hormonal regulation of glucokinase gene expression was investigsted in cultured rat islet cells. To measure glucokinase mRNA in pancreatic islet cells, the competitive PCR method was adopted. With this method, GKmRNA levels can be measured using only 0.1-1.0 microgram of total RNA isolated from cultured rat islet cells. Following 24 h preculture with 5.5 mM glucose, islet cells were cultured for 24 or 8 h with hormonal or non-hormonal factors. Glucokinase mRNA levels tended to increase, but not significantly, at 16.7 mM glucose compared to those at 5.5 mM glucose. Treatment with either 1 microM T3 or 1 microM glucagon resulted in a decrease in the glucokinase mRNA level with 16.7 mM glucose, whereas 1 microM insulin had no effect on glucokinase mRNA. Five mM dibutyryl cyclic AMP decreased the glucokinase mRNA level with 16.7 mM glucose, but cycloheximide did not block this inhibitory effect, suggesting that the effect of glucagon may be mediated by cyclic AMP and that protein synthesis is not involved in the response. Furthermore, the islet glucokinase mRNA level increased in response to 1 microM glibenclamide with 5.5 mM glucose and the response was abolished by cycloheximide, which indicates the involvement of protein synthesis in the glibenclamide-induced mRNA change. An 8-bromo-cyclic GMP (1 microM) and vanadate (1 microM) did not affect the islet GKmRNA level. These findings suggested that thyroid hormone and glucagon-cyclic AMP suppress, and glibenclamide increases the GKmRNA level in cultured rat islet cells, and that insulin, cyclic GMP and vanadate differentially affect glucokinase gene expression in pancreatic islet cells and in the liver.

ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case.

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half-life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.

Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

OBJECTIVE: The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. METHODS: We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. RESULTS: RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. CONCLUSION: We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.

Dynamics of Ca2+ transients in norepinephrine-stimulated individual H-35 hepatoma cells: fura-2 digital imaging microscopy and high time-resolution microspectrofluorometry.

We investigated spatiotemporal changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10(-6) M) resulted in an initial transient increase in [Ca2+]i, followed by a small sustained [Ca2+]i plateau above the pre-stimulation level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an alpha-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca(2+)-free medium or by pre-treatment of cells with La3+. On the other hand, the sustained plateau was eliminated by Ca(2+)-free medium or La3+. Therefore, H-35 cells have a Ca(2+)-signaling pathway which is activated via alpha-adrenergic receptors. Mn2+ entered the cytosol after NE stimulation, as shown by quenching of fura-2. This indicates that H-35 hepatoma cells possess Mn(2+)-permeable Ca2+ channels at the plasma membrane. In addition, the Ca2+ efflux pattern from H-35 cells to the extracellular space during NE stimulation was visualized by digital imaging microscopy when free fura-2 was equilibrated between the cells and the extracellular space. The efflux of Ca2+ from H-35 begins between the initial [Ca2+]i transient and the sustained [Ca2+]i plateau.

Ca2+ dynamics in rat pancreatic AR-42J and AR-IP cells.

Spatiotemporal change of the cytosolic free Ca2+ concentration ([Ca2+]i) in response to a variety of secretagogues was examined in rat pancreatoma AR-42J and AR-IP cells by microspectroflurometry and digital imaging microscopy after loading with fura-2. In the presence of external Ca2+, carbachol, CCK-OP (cholecystokinin-octapeptide), gastrin, norepinephrine or high K+ evoked a large transient increase in [Ca2+]i in AR-42J cells which declined to a sustained level before slowly declining towards the resting level. In the absence of external Ca2+, a transient increase in [Ca2+]i were evoked by all the ligands except for high K+ stimulation, which declined rapidly towards the resting level. The [Ca2+]i increase caused by carbachol and high K+ treatment was inhibited by muscarinic receptor antagonist, atropine, and by L-type Ca2+ channel blocker, nifedipine, respectively. The transient [Ca2+]i increase induced by gastrin stimulation was not blocked by Ca2+ channel blocker, lanthanum. In the AR-IP cells, which are non-differentiated pancreatoma cell line, all stimulations including high K+ treatment have failed to evoke [Ca2+]i response. These intracellular Ca2+ mobilizations in response to ligands in AR-42J cells were displayed by digital imaging microscopy. From these results we conclude that AR-42J cells has an alpha-adrenergic receptor, in addition to muscarinic acetylcholine receptor, CCK-OP receptor, gastrin receptor and voltage dependent Ca2+ channel. In marked contrast, AR-IP cells have neither any hormone receptor for the above ligands nor voltage dependent Ca2+ channel.

Chemical diagnosis of Fabry's disease by fluorometric assay and fast atom bombardment/mass spectrometry.

We report the results of a fluorometric assay for alpha-galactosidase A (EC.3.2.1.22) in plasma and leukocytes, and fast atom bombardment/mass spectrometry (FAB/MS) analysis of glycosphingolipids in urine sediments from a patient with Fabry's disease. In plasma, this patient had only 5.0% of the normal amount of alpha-galactosidase A, and his brother and mother had 11.0% and 25.0%, respectively. In leukocytes, the activities were below 8.0%. Glycosphingolipids from urine sediments were partially purified using a Sep-Pack C18 cartridge. The chemical diagnosis of Fabry's disease can be made more rapidly and accurately using fluorometric and FAB/MS analyses.

Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene.

cDNA clones for serine:pyruvate aminotransferase (SPT, alternative name: alanine:glyoxylate aminotransferase) were obtained from a cDNA library constructed from the liver of a primary hyperoxaluria type I (PH1) case in which the SPT activity was approximately one-hundredth that in control liver. Six clones were isolated from 100,000 transformants and all of them contained an approximately 1.5 kbp insert which included the whole coding region for human SPT. Nucleotide sequence analysis revealed a point mutation of T to C at position 634 (relative to the 5'-end of the cDNA) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1.

Generation from a single gene of two mRNAs that encode the mitochondrial and peroxisomal serine:pyruvate aminotransferase of rat liver.

In rat liver there are two types of serine:pyruvate aminotransferase (SPT) whose natures are indistinguishable but whose subcellular localization are different. One is a mitochondrial enzyme (SPTm) and the other a peroxisomal enzyme (SPTp). We compared, in this study, the structure of mRNAs encoding SPTm and SPTp by comparison of the sizes after removal of poly(A) tail by ribonuclease H and by means of RNA blot analysis and S1 nuclease protection assay. No differences were detected between these two mRNAs other than that about 100 nucleotides of the 5'-terminal sequence of SPTm mRNA are lacking in SPTp mRNA, and the length of the poly(A) tail is different. Southern blot analysis of rat genomic DNA showed that the SPT gene is single. Primer extension and S1 nuclease mapping analyses, using a DNA fragment of a genomic clone, revealed that the SPTm and SPTp mRNAs are transcribed from different initiation sites, about 70 nucleotides apart, in the same exon, exon 1. Ribonuclease protection assay performed with RNA hybridization probe corresponding to 5'-terminal portion of SPTm mRNA also showed that the 5'-terminal sequence of SPTp mRNA is about 70 nucleotides shorter than that of hormone-responsive SPTm mRNA. These results indicate that the different organelle distribution of SPTm and SPTp, the products of the same SPT gene, arises from transcription from different initiation sites, conferring N-terminal extension peptide, the mitochondrial targeting signal, only on the translation product of SPTm mRNA.

[A case of adult Still's disease with pulmonary hypertension].

The patient was a 29-year-old woman. She was well until autumn 1983, when she presented with polyarthralgia, fever above 39 degrees C, hepatosplenomegaly, swelling of lymphnode and salmon pink rash. Laboratory tests revealed marked leucocytosis with shift to the left, elevated ESR, strong positivity of CRP and abnormal liver function tests. However, anti-nuclear antibody and RA factor were negative. She was diagnosed as adult onset Still's disease (AOSD) by characteristic clinical course and laboratory data. During her disease course these abnormal findings could be well controlled neither by nonsteroidal anti-inflammatory drugs, immunosuppressive agents nor corticosteroids. Two and half years after the first admission, she began to complain of dry cough, dyspnea on efforts. Auscultation revealed an increased pulmonic sound and systolic murmur of cardiac apex. Chest X-Rays showed enlarged main pulmonary arteries. The lung fields were normal. Pulmonary function tests gave no evidence of a significant obstructive or restrictive defect but showed the low DLco and hypoxemia. Ventilation-perfusion lung scanning failed to reveal pulmonary embolism. Finally, right heart catheterization confirmed the pulmonary precapillary hypertension. Her pulmonary hypertension has progressed rapidly, strongly suggesting poor prognosis. Her pulmonary hypertension associated with no apparent parenchymal involvement was thought to be caused by a pulmonary vascular change probably related to AOSD. This case is a first case of AOSD with pulmonary hypertension.

[A case of overlap syndrome of PSS, SLE and Sjögren syndrome treated by cyclosporin A: an improvement of sclerosis of the skin].

A 26-year-old female patient complicated with PSS, SLE and Sjögren was treated by combination of medium doses of corticosteroid and cyclosporin A. On admission, she showed many abnormal laboratory findings such as increased serum gammaglobulin level, high anti-DNA antibody titer and low compliment level. This therapy improved not only these laboratory abnormalities but clinical symptoms including sclerosis of the skin and arthritis, without notable side effect due to either of these drugs for the following 10 months so far. On the other hand we haven't acknowledged the effect to internal involvement yet. Since there is no promising therapy to sclerosis of the PSS skin, it is suggested that cyclosporin A can be an effective agent for PSS and should be further evaluated.

Absorption of egg antigens by the gut observed by oral Prausnitz-Küstner (Walzer) reaction in atopic dermatitis.

Sera from 50 children (27 boys and 23 girls, under the age of 3 years) with atopic dermatitis allergic or not to hen's egg shown by skin test or radioallergosorbent test (RAST) were passively transferred to recipients which were then challenged with injection of egg antigen (Prausnitz-Küstner (P-K) test) or with ingestion of a raw egg (oral P-K test). Thirty-one patients showed positive P-K reaction with serum titers from 2 to 8,192. Fifteen of the P-K positive cases were also positive in the oral P-K test with titers from 2 to 256. The ratio of the oral P-K titer and the P-K titer in each positive case was from 1:2 to 1:32. The results indicate that a high percentage of atopic dermatitis patients with egg allergy have IgE antibody in the serum capable of reacting with an ingested egg.