Responding to hypoxia: lessons from a model cell line.

Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca2+-calmodulin pathway, the 3'-5' adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O2-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O2, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells.

Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase (SH-EP)

A plant cysteine endopeptidase, designated SH-EP, is a major protease occurring in cotyledons of Vigna mungo seedlings, and acts to degrade seed globulin stored in protein bodies. Here we show that the 43 kDa intermediate of SH-EP formed in the endoplasmic reticulum is transported to protein bodies and processed to the 33 kDa mature form during transport or thereafter, and that the COOH-terminal propeptide of 10 amino acid residues containing a KDEL sequence, which is known as a retention signal for the endoplasmic reticulum lumen, is processed to form the mature SH-EP.

Cold water swim stress increases the expression of neurotensin mRNA in the lateral hypothalamus and medial preoptic regions of the rat brain.

Stress-induced analgesia is a well-documented phenomenon that occurs in all mammalian species. Forced cold water swim produces a type of stress-induced analgesia that is independent of mu opioid receptors. The neuropeptide neurotensin (NT) has been implicated in mu opioid-independent analgesia (MOIA), but the circuitry of this system is largely unknown. The medial preoptic area (MPO) and lateral hypothalamus (LH) are two regions that are known to modulate pain processing. These two regions also contain neurotensinergic projections to the periaqueductal gray, a region that has been shown to produce MOIA upon injection of NT. The goal of this study was to determine if cold water swim (CWS) stress, which produces MOIA, activates the NT-ergic systems in these two regions. In situ hybridization results indicate that CWS increases the level of NT mRNA within neurons in the MPO and LH, suggesting that these two regions are activated during this process.

Insulin degradation by Madin-Darby canine kidney cells expressing the insulin receptor.

Prior studies have shown that Madin-Darby canine kidney cells (MDCK) overexpressing the human insulin receptor bind and respond normally to insulin (T.C. Yeh, R.A. Roth, Diabetes 43 (1994) 1297-1303). Moreover, the insulin receptor preferentially localizes to the basolateral membrane of these cells. In the present studies, insulin was added to either the apical or the basolateral side of these cells and the extent of degradation of the insulin was assessed. Radioactive insulin added to either side was bound to its receptor and the radioactivity which reached the other side of the cell was to a large extent degraded fragments. Insulin added to the apical side was degraded to a larger extent (83%) than when added to the basolateral side (49%) although the basolateral side has much more insulin receptors than the apical side. This degradation process was not inhibitors of either lysosomal enzymes, the proteasome complex or cathepsins. The degradation process could however, be potently inhibited by the sulfhydryl alkylating agent N-ethylmaleimide. Further, cell surface biotinylation study showed that the insulin degrading enzyme was preferentially localized on the apical membranes. These results suggest that insulin added on the apical side of MDCK cells are more closely linked to the degradation process than that added on the basolateral side.

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Application to cerebellar protein mapping.

A method is presented for the structural characterization of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 x 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.

The molecular basis of O2-sensing and hypoxia tolerance in pheochromocytoma cells.

Hypoxia is a common environmental stimulus. However, very little is known about the mechanisms by which cells sense and respond to changes in oxygen. Our laboratory has utilized the PC12 cell line in order to study the biophysical and molecular response to hypoxia. The current review summarizes our results. We demonstrate that the O2-sensitive K(+) channel, Kv1.2, is present in PC12 cells and plays a critical role in the hypoxia-induced depolarization of PC12 cells. Previous studies have shown that PC12 cells secrete a variety of autocrine/paracrine factors, including dopamine, norepinephrine, and adenosine during hypoxia. We investigated the mechanisms by which adenosine modulates cell function and the effect of chronic hypoxia on this modulation. Finally, we present results identifying the mitogen- and stress-activated protein kinases (MAPKs and SAPKs) as hypoxia-regulated protein kinases. Specifically, we show that p38 and an isoform, p38gamma, are activated by hypoxia. In addition, our results demonstrate that the p42/p44 MAPK protein kinases are activated by hypoxia. We further show that p42/p44 MAPK is critical for the hypoxia-induced transactivation of endothelial PAS-domain protein 1 (EPAS1), a hypoxia-inducible transcription factor. Together, these results provide greater insight into the mechanisms by which cells sense and adapt to hypoxia.

Thyroid carcinoma.

Pathological and clinical characteristics of thyroid carcinoma are reported. In 379 routine consecutive autopsies in which carcinoma was not suspected, serial sections of thyroid gland were histologically examined. Latent thyroid carcinoma was detected in 15.7% of the glands. Papillary adenocarcinoma comprised 76.3%, follicular adenocarcinoma 22.0% and trabecular carcinoma 1.7% of the cases. Sclerosing carcinoma was seen in 32 cases. The long diameter of 90% of these tumors was less than 5 mm. Whether or not thyroid carcinoma persists as such a small carcinomatous lesion indefinitely was studied along with its clinical significance. Carcinomatous lesions complicating hyperthyroidism were always less than 1 cm. About 70% were less than 5 mm long. Since proliferation of some thyroid carcinomas was inhibited by TSH suppression therapy, growth and proliferation of carcinoma in the presence of excessive thyroid hormone or hyperthyroidism is probably already inhibited.

[Effect of LDL-apheresis on a case of Cerebrotendinous Xanthomatosis].

Cerebrotendinous Xanthomatosis (CTX) is a rare familial disease characterized by tendon-xanthomas, cataracts, progressive cerebellar ataxia, dementia and an elevation of serum cholestanol with normal levels of cholesterol. Although the pathogenesis of CTX is not fully understood, increment of cholestanol is suggested one of the major metabolic derangements of the disease. Recently, the LDL-apheresis has been developed as a new therapeutical equipment in the field of hyperlipidemia and been widely used to reduce the levels of LDL-cholesterol by selective LDL adsorption. From the point of view that cholestanol is involved mainly in LDL-cholesterol (1.019 less than d less than 1.063), we used this LDL-apheresis in the aim of reducing the cholestanol in 58 years old woman with typical sign and symptoms of CTX. The levels of serum cholestanol and cholesterol before the treatment with LDL-apheresis, were 10.7 micrograms/ml and 175 mg/dl respectively. Also the ratio of cholestanol/cholesterol indicated 0.63. By the first procedure of apheresis, the level of cholestanol was markedly decreased to 5.2 micrograms/ml (50%). Several LDL-apheresis treatments were carried out once a month. During 5 months treatments, neurological deterioration was arrested, dementia which included disorientation and recent-memory loss, cleaned a little. Although the xanthomas did not decrease in size, this patients was better oriented to person, place, time and was able to speak rationally, 2nd her cerebellar dysfunction revealed improvement. From our new experiments-we believe that the LDL-apheresis offers the strong hope of preventing the progress on cerebrotendinous xanthomatosis.

[Peripheral blood stem cell transplantation for rapidly spreading myeloma].

We report a case of rapidly spreading myeloma of immature cell morphology treated by peripheral blood stem cell transplantation (PBSCT). A 54-year-old man had a right orbital tumor, which subsequently was removed and proved to be plasmacytoma. Three years later a mass lesion appeared in his left lung and bilateral kidneys. The specimen obtained at lung biopsy confirmed the diagnosis of plasmacytoma. Serum M-protein, IgG lambda was increased, but there was no increase in plasma cells in the bone marrow. Since chemotherapy with VAD did not show any improvement, a high dose etoposide (500 mg/day, 4 days) was administered. When bone marrow suppression recovered, PBSCs were harvested (3.3 x 10(6)/kg). After conditioning therapy with cyclophosphamide (2.0 g/day, 2 days), etoposide (200 mg/day, 3 days) and ranimustine (200 mg/day, 2 days), the stored PBSCs were injected. Minor response was obtained and he was discharged. 2 months thereafter, it was found that plasma cells increased in the bone marrow. He died of pulmonary bleeding soon. Autopsy revealed immature plasma cell infiltration in multiple organs including the heart, liver, spleen, kidneys, intestine, bone and bone marrow.

[Immunohistochemical study of p53 and Ki-67 overexpression in grade 3 superficial bladder tumor in relationship to tumor recurrence and prognosis].

PURPOSE: The major drawback of the current treatment for superficial bladder tumor is the high rate of recurrence. Especially, the tumor with grade 3 component has a tendency to recur and progress in stage. However, we have difficulty in predicting tumor recurrence and stage progression accurately by conventional clinicopathological factors. We evaluated the efficacy of p53 and Ki-67 overexpression as a predictor of recurrence or prognosis in patients with superficial bladder tumor of grade 3. MATERIALS AND METHODS: Samples were obtained from 41 patients with superficial transitional cell carcinoma of the bladder of grade 3 who were treated by transurethral resection (TUR). The immunohistochemical study was performed using the antibodies against the p53 protein and Ki-67 antigen on formalin-fixed, paraffinembedded tissue specimens from initial tumors. We evaluated the correlation between these results and several clinicopathological factors. RESULTS: The p53 index and the Ki-67 index in pTa, pT1a and pT1b tumors were 26.4 +/- 30.1%, 28.6 +/- 30.0%, and 34.6 +/- 32.6% (p53) and 20.5 +/- 22.5%, 20.0 +/- 29.3%, and 29.2 +/- 28.4% (Ki-67). There was no significant difference between the each index and tumor stage. Eighteen cases (43.9%) had intravesical recurrence. The p53 index of the initial tumor from the tumor free cases (n = 23), recurrent cases without stage progression (n = 12), and stage progression cases (n = 6) were 19.7 +/- 28.2%, 42.0 +/- 28.7%, and 42.5 +/- 32.0%. Between the recurrence-free cases and the recurrent cases without progression, the p53 index of the initial tumor had statistical significance (p < 0.05). The Ki-67 index was shown to be the same pattern as the p53 index, but there was not statistical significance. Four of patients with stage progression had tumor progression within six months. Three of the patients with tumors with stage progression died of the cancer. In multivariate analysis, tumor multiplicity (p = 0.01), BCG intravesical instillation (p = 0.04), p53 index (p = 0.01) and Ki-67 index (p = 0.02) were the positive risk factors for tumor recurrence, but only the p53 index was the positive risk factor for prognosis fo the patients (p = 0.03). CONCLUSION: These results suggest that the immunohistochemical study of p53 overexpression is a useful predictor for tumor recurrence and prognosis in patients with superficial bladder tumor with grade 3.

[Chinese herbs nephropathy in the Kansai area: a warning report].

In 1993, Vanherweghem and his associates reported cases of rapidly progressive renal interstitial fibrosis in young women who were administered a slimming regimen including Chinese herbs. Subsequently, similar cases have been reported. In Japan, especially in the Kansai area, several cases of Chinese herbs nephropathy have already been reported. We experienced a patient suffering from Chinese herbs nephropathy (CHN), and further detected aristolochic acids from the Chinese herbs taken by the patient. Aristolochic acids are known to be causative agents of CHN. The danger of CHN should be noted as soon as possible and drugs containing aristolochic acids should be prohibited.

[Traditional remedy-induced Chinese herbs nephropathy showing rapid deterioration of renal function].

A 19-year-old female was referred to our hospital for azotemia and anemia. She had been taking a health food for atopic dermatitis for about three years. Urinalysis showed proteinuria, glycosuria and microscopic hematuria. Generalized aminoaciduria was observed. Moreover, severe anemia, azotemia, hypokalemia and hypophosphatemia were also observed. Renal biopsy specimen disclosed hypocellular interstitial fibrosis and degeneration of the proximal tubular epithelial cells. No remarkable changes were observed in the glomeruli. Aristolochic acid was detected in the health food. From these findings, she was diagnosed as having Chinese herbs nephropathy (CHN). Although consumption of the food intake was stopped, her renal function deteriorated rapidly. Previously, we reported that certain kinds of Chinese herbal drugs contain aristolochic acid and that the drugs should be prohibited if aristolochic acid is identified. However, we experienced a patient of CHN arising from traditional remedy, which was not proved to be safe. It should be awared that health foods may contain aristolochic acid.

[An autopsy case of corticobasal degeneration clinically misdiagnosed as Pick's disease].

We report a 69-year-old woman who was clinically diagnosed as having a frontal lobe-type of Pick's disease. The initial symptoms were personality changes and problematic behaviors. The patient showed intellectual decline, "stehende Redensarten" and abnormal attitude in interpersonal situations such as inattentiveness and indifference in the course of the disease. Brain CT revealed a marked atrophy of the frontal lobes. In the terminal stage the patient had severe dementia, mutism, parkinsonism and cervical dystonia. Neuropathologically, there was a marked atrophy of the frontal lobes. The superior frontal gyrus was most severely atrophic. Histological study revealed mild to moderate loss of neurons, hyperplasia of protoplasmic astrocytes and many balooned neurons in the deep layers of the atrophied cerebral cortex. Severe neuronal loss was even seen only in a part of the superior frontal gyrus. The cerebral white manner showed marked diffuse fibrillary gliosis. There was neuronal loss with gliosis in the thalamus, lentiform nucleus, subthalamic nucleus, substantia nigra and inferior olivary nucleus. Marked gliosis was seen in the midbrain and pontine tegmentum. Sections from several levels of the spinal cord also showed marked gliosis of the gray matter. Antibodies against human tau stained massive argyrophilic thread-like structures and oligodendroglial microtubular masses in the affected lesions. Neurofibrillary tangles were localized in the hippocampus and parahippocampal region. Neither Pick's body nor senile plaque were observed. Corticobasal degeneration (CBD) is a neurodegenerative disease initially presenting with unilateral motor disturbances. Typical initial symptoms are rigidity, akinesia and apraxia of an affected arm. The clinical phenotype might depend upon the affected areas of the cerebral cortex. Our patient initially exhibited personality changes and was clinically diagnosed as having Pick's disease. Although our case had unusual distribution pattern of the cerebral atrophy, it was pathologically diagnosed as CBD. The review of the literature suggests the presence of clinical varieties in CBD.