Functional evidence for a telomerase repressor gene on human chromosome 10p15.1.

Based on the sites of frequent allelic loss in hepatocellular carcinoma, five normal human chromosomes (2, 4, 5, 10 and 16) were transferred individually into a telomerase-positive human hepatocellular carcinoma cell line, Li7HM, by microcell-mediated chromosome transfer (MMCT). Chromosome 10, but not the others, repressed telomerase activity immediately and stopped cell growth after 50 population doublings (PDs). Loss of the transferred 10p loci resulted in the emergence of revertant cells that continued to proliferate and expressed telomerase activity, suggesting the presence of a telomerase repressor gene on this chromosomal arm. Transfer of a series of defined fragments from chromosome 10p successfully narrowed down the responsible region: a 28.9-cM region on 10p15 (between WI-4752 and D10S249), but not a 26.2-cM region (between D10S1728 and D10S249), caused repression of telomerase activity and progressive telomere shortening. A strong correlation between the expression level of telomerase catalytic subunit gene (hTERT) and telomerase activity was observed. These findings suggest that a novel telomerase repressor gene which controls the expression of hTERT is located on the 2.7-cM region (between WI-4752 and D10S1728) on chromosome 10p15.1.

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles.

Although the enzyme (Na+ + K+)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na+ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na+ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na+ transport sensitively. This technique relies on the presence of Na+-Ca2+ exchange and ATP-driven Na+ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na+ uptake is monitored by a subsequent Nai+-dependent Ca2+ uptake reaction (Na+-Ca2+ exchange) using 45Ca2+. We present evidence that the Na+-Ca2+ exchange will be linearly related to the prior active Na+ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na+ isotopes. Applying this method, we measure cardiac ATP-dependent Na+ transport and (Na+ + K+)-ATPase activities in identical ionic media. We find that the (Na+ + K+)-ATPase and the Na+ pump have identical dependencies on both Na+ and ATP. The dependence on [Na+] is sigmoidal, with a Hill coefficient of 2.8. Na+ pumping is half-maximal at [Na+] = 9 mM. The Km for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na+ pumping. This approach should allow other new investigations on ATP-dependent Na+ transport across cardiac sarcolemma.

Sodium-calcium exchange and sidedness of isolated cardiac sarcolemmal vesicles.

The sidedness of isolated rabbit cardiac sarcolemmal vesicles was studied by observing the effects of several permeability-increasing agents on measurements of the amount of sialic acid released by neuraminidase, specific ouabain binding, and K+-phosphatase and (Na+ + K+)-ATPase activities. The results suggest that the vesicles are sealed and are about 80% right-side-out. Na+-Ca2+ exchange exhibited by these vesicles could be attributed to the sarcolemma rather than to some contaminating organelle. Ca2+ uptake was stimulated by preloading the vesicles with NaCl (and not KCl). Increasing the external [Na+] induced a rapid Ca2+ loss, which could not be mimicked by K+, Li+, Rb+ or choline+. The Nai+-dependent Ca2+ uptake was inhibited by certain cations: Cd2+ > La3+ > Y3+ > Mn2+ > Co2+ > Mg2+. The Nai+-dependent Ca2+ influx was enhanced by an inside positive membrane potential and inhibited by an inside negative membrane potential. Potentials were induced by a K+-valinomycin system.

Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis.

Telomeres shorten progressively with age in normal somatic cells in culture and in vivo. The maintenance of telomere length is assumed to be an obligatory step in the progression and immortalization of most human tumor cells. To understand the role of telomere dynamics in the development of hepatocellular carcinoma (HCC), we examined the length of terminal restriction fragment (TRF), as an indicator for telomere length, in HCC and surrounding tissues with chronic active hepatitis (CAH) or liver cirrhosis (LC). The study was performed in 12 hepatitis C virus (HCV) antibody-positive, 12 hepatitis B virus (HBV) antigen-positive tissues, and 4 tissue samples from virus-negative patients with HCC. The peak TRFs in all 3 types of HCC were significantly shorter than those of the surrounding tissues (i.e., LC or CAH). TRFs examined in one patient with atypical adenomatous hyperplasia (AAH) also was shortened. Thus, progressive TRF shortening occurs from normal to CAH to LC to HCC(AAH). Telomerase, an enzyme that adds repeated telomere sequences onto the chromosome ends and stabilizes telomere length in immortal cells, also was examined in tissues and detected in high levels almost exclusively in HCCs. Interestingly, the intensity of telomerase activity in the AAH case was similar to that of HCC. In addition, the telomerase activity of biopsy samples with a fine 21-gauge needle also was examined in 10 HCCs, 2 adenomatous hyperplasias (AHs), 2 LCs, and 2 CAHs. We found strong telomerase activity in all the HCCs and surprisingly in the 2 cases that were pathologically diagnosed as AH. Thus, the findings strongly suggest that persistent cell proliferation or rapid cell turnover through damage of hepatic cells result in a process of multistep hepatocellular carcinogenesis. Thus, progressive shortening of telomeres and the activation of telomerase may be a useful marker for the early detection of malignant progression in liver disease.

Reverse cellular distribution of calmodulin to S-100 protein in primate brain.

Both calmodulin and S-100 protein are Ca2+-binding proteins of the EF-hand family. Immunocytochemical study revealed that calmodulin existed mainly in the neurons, whereas S-100 protein was localized primarily in the glial cells of the cerebral and cerebellar cortices of man and monkey. The observed reverse cellular distribution of calmodulin to S-100 protein in primate brain suggests that calmodulin might be replaced in its role as a Ca2+-binding protein by S-100 protein in the glial cells.

Intraparenchymal allografts in the mouse brain in relation to immunocytochemical identification of T lymphocyte subsets.

In a study of intracerebellar allografts of mice brainstem anlagen (embryonic day 12-14), we examined immunocytochemically the expression of two different types of T lymphocytes in and around the grafts. Helper/inducer and cytotoxic/suppressor T cells were identified with anti-L3T4 and anti-Lyt-2 monoclonal antibodies, respectively. Allografts into major histocompatibility complex (MHC)-compatible recipients showed no histological signs of rejection such as marked neovascularization and cellular infiltrates even 6 months after transplantation, but those into MHC-incompatible recipients generally had rejection reactions within one month after transplantation. In the latter cases, the L3T4/Lyt-2 ratio for the T lymphocytes in the infiltrates of the grafts was 1.03 +/- 0.14 (mean +/- S.D.), suggesting that both helper/inducer and cytotoxic/suppressor T cells may play important roles in the mediation of intraparenchymal brain allograft rejection.

Effects of cyclic AMP on S-100 protein level in C-6 glioma cells.

Dibutyryl cyclic AMP (dbcAMP) markedly elevated S-100 protein level in C-6 glioma cells in vitro. Quantitative analysis by two-dimensional polyacrylamide gel electrophoresis revealed that the elevation was caused by a combination of increased synthesis and reduced degradation of S-100 protein in C-6 cells exposed to dbcAMP. These results suggest that dbcAMP affects both the synthesis and the degradation of S-100 protein in C-6 cells.

Evaluation of the drug-induced morphological differentiation of rat glioma cells (C-6) from the aspects of S-100 protein level and con A binding pattern.

The intracellular content of the nervous system specific protein S-100 began to increase with 4 days latency following the morphological differentiation of cultured rat glioma cells (C-6) with 1 mM dibutyryl cyclic AMP (dbcAMP), rising to approximately 10-fold over the control level at 15 days after the treatment. The concanavalin A (Con A) binding pattern on the external cell surface of C-6 cells exposed to dbcAMP appeared as a smooth layer of 40-60 nm thickness whereas that of control cells was irregularly thick and patchy. The correlation between morphological and biochemical changes of C-6 cells after dbcAMP treatment is discussed in relation to the mechanism controlling the differentiation of glioma cells.

Hyperthermia for brain tumors: improved delivery with a new cooling system.

Hyperthermia has emerged as an adjunct to other forms of brain tumor therapy. Interstitial microwave irradiation is an effective method of inducing localized brain hyperthermia. One of the problems with this technique, however, is the overheating of tissue adjacent to the antenna. In this study, a cooling system for the interstitial microwave antenna was developed for the purpose of providing uniform and accurate heating by the elimination of overheating. The ability to generate more uniform hyperthermic fields was evaluated in normal monkey brains. Six monkeys under general anesthesia and controlled respiration underwent parietooccipital craniectomies 4 x 4 cm in size. The antenna cooling system was constructed of a silicone tube 5.0 mm in outer diameter. Silicone-coated interstitial microwave antennae 1.5 mm in diameter were used. A single antenna or a square array (1.6 cm on a side) of 4 antennae was inserted into the brain with the coupled system to a depth of 2 cm. The brain tissue was heated by 2450-MHz microwave irradiation. Temperature distributions were mapped using nonperturbing thermocouples. These thermal profiles were compared with those generated without the cooling system. In the experiments with the single antenna, the antenna cooling system eliminated the overheating and rapid radial falloff in temperature, without a reduction of the hyperthermic field. In the four-antennae experiments using the cooling system, the thermal field was dramatically flattened with minimal reduction in size; however, the area maintained at a therapeutic temperature range (42-45 degrees C) was significantly enlarged by the cooling system.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemifacial spasm caused by a spontaneous dissecting aneurysm of the vertebral artery. Case report.

The authors describe the first reported case of dissecting aneurysm presenting with hemifacial spasm. The patient was a 58-year-old woman with left hemifacial spasm of 2 years' duration. Cranial nerve examination was otherwise normal and no other clinical symptoms were observed. Vertebral angiography revealed a fusiform enlargement of the left vertebral artery and contrast medium remaining in the intramural false lumen in the venous phase. Microvascular decompression of the facial nerve with wrapping of the aneurysm resulted in complete relief of the hemifacial spasm.

Primary intracranial choriocarcinoma.

The authors report four cases of primary intracranial choriocarcinoma, all in young males. There tumors occurred in the pineal region and one in the lateral ventricle. Besides the signs of increased intracranial pressure and ophthalmological disorders, skin pigmentation or eruption was observed in three cases and precocious puberty in two cases. Angiography revealed tumor stain with irregular vessels in all cases. Enhanced computerized tomography scans in the last two cases demonstrated a round, lobulated lesion of high density, with relatively low density in the central portion. Hormonal study was carried out in three cases. In addition to high levels of human chorionic gonadotropin (HCG) in the urine and cerebrospinal fluid and/or of plasma luteinizing hormone (LH), LH in tumor tissue or medium obtained via cell cultures was also high in all three cases, suggesting inherent activity of HCG secretion by the tumor cells. The tumors were relatively demarcated from the surrounding brain. They were very hemorrhagic, and fresh hematoma was identified in the tumor in case. Good results were obtained in two cases treated with surgical removal followed by simultaneous chemotherapy and 60Co irradiation.

Primary central nervous system lymphoma.

A retrospective analysis of 21 cases of primary central nervous system (CNS) lymphoma is reported. All patients presented with a solitary mass in the supratentorial region. None had previously received immunosuppressive therapy. Neuroradiological studies included technetium-99m-pertechnetate brain scanning in eight cases, cerebral arteriography in all 21 cases, and computerized tomography (CT) in 14 cases. The characteristic features were increased uptake in brain scans, mass effect in arteriograms, and marked contrast enhancement on CT scans. Abnormal tumor vessels were occasionally seen on arteriography, and subtraction films were usually required to appreciate tumor stain. All patients underwent craniotomy, and histological studies of the tumors showed a diffuse type of lymphoma in all cases. Immunoglobulin testing was performed in 19 cases and a monoclonal spike was verified in 10, suggesting a B cell origin. All patients were followed until their death except one who was still alive 12 months from onset of symptoms. Therapy included subtotal resection in all 21 cases, whole-brain irradiation in six cases, chemotherapy in two cases, and a combination of whole-brain irradiation and chemotherapy in nine cases. Three different forms of chemotherapy were used. The results suggest that chemotherapy is an important addition to subtotal resection and whole-brain irradiation in the treatment of primary CNS lymphoma.

Direct thrombosis of aneurysms with cellulose acetate polymer. Part II: Preliminary clinical experience.

The authors report the treatment of seven intracranial aneurysms in six patients with direct infusion of cellulose acetate polymer solution, a new liquid thrombotic material. These aneurysms were considered inoperable because of their size or location, or because of the patient's neurological condition. This material avoids the difficulties associated with balloon occlusion, and completely fills even irregularly shaped aneurysms. Cellulose acetate polymer solution hardens in about 5 minutes and remains solid once inside the aneurysm. Because this technique is less invasive than surgery, it can be used for high-risk patients in the acute stage of subarachnoid hemorrhage. Transient motor aphasia occurred in one patient. A small residual neck, which caused rebleeding 3 months after the treatment, remained in another patient. This article describes the new material, the procedure for direct thrombosis, and preliminary clinical results.

Anterior inferior cerebellar artery aneurysm. Report of three cases.

Three cases of aneurysms of the anterior inferior cerebellar artery are reported. Two of the aneurysms were located in the cerebellopontine angle and one in the ventral portion of the pons. Through a suboccipital craniectomy, beck clipping was performed on one aneurysm, neck ligation on another, and coating on the third. A discussion of the surgical procedures and complications includes a review of previous reports.

Acute intracranial hypertension and brain-stem blood flow. An experimental study.

This study has been carried out to evaluate the effect of supratentorial mass lesions on the local cerebral blood flow (CBF) of the brain stem. Local CBF of the thalamus, inferior colliculus, and medulla oblongata, and supra- and infratentorial pressure were serially measured in 52 cats with intracranial hypertension produced by supratentorial balloon expansion. The mean control local CBF's in the thalamus, inferior colliculus, and medulla oblongata were 37.5, 42.1, and 30.7 ml/100 gm/min, respectively. At 20 to 30 mm Hg of supratentorial pressure, the local CBF of the thalamus started to decrease, and at 20 mm Hg of infratentorial pressure, the local CBF of the inferior colliculus began to decrease. Finally, at 40 to 60 mm Hg of infratentorial pressure, the local CBF of the medulla oblongata was affected. At the beginning of uncal herniation, indicated by anisocoria, the mean local CBF of the inferior colliculus abruptly decreased from 33.7 to 19.6 ml/100 gm/min in 16 cats. The Cushing response was evoked at a mean supratentorial pressure of 93.4 mm Hg and infratentorial pressure of 49.9 mm Hg in 16 cats. When the systemic arterial pressure was increased to the highest level in 13 cats, the mean local CBF of the medulla oblongata did not show significant change (a decrease from 22.8 to 20.9 ml/100 gm/min). The results suggest that at the beginning of uncal herniation, the local CBF of the upper brain stem markedly decreased. During the Cushing response, the local CBF of the medulla oblongata did not change significantly.

Sensory and motor responses to deep brain stimulation. Correlation with anatomical structures.

Motor and sensory responses induced by trial stimulation were examined before stereotaxically implanting a permanent stimulating electrode for pain relief in 11 patients with intractable pain of central origin. The total number of points eliciting a response when stimulated was 70. The points of stimulation were determined as exactly as possible from Schaltenbrand and Bailey's Atlas. Motor responses were detected upon stimulating 21 points, the majority of which were in the posterior third of the posterior limb of the internal capsule (IC). Stimulation of these 21 points was accompanied by pain relief in only two points (10%). Warm (22) or cool sensations (three) were provoked in the most posteromedial portion of the posterior limb of the IC, nucleus reticularis pulvinaris, and area triangularis, and seven (28%) of these 25 sensations were accompanied by pain relief. A burning sensation was found upon stimulation of 12 points, with stimulation in the mesencephalic lateral tegmental field eliciting the most severe burning pain. A tingling sensation was elicited at 12 points, in a distribution similar to that of the warm sensation. Five (42%) of these 12 points provided pain relief. The best stimulating point for pain relief is not in the center of the posterior limb of the IC, directly lateral to the posterior commissure, but rather in its most posteromedial part; that is, at the nucleus reticularis pulvinaris or area triangularis.

Establishment of a brain-tumor model in adult monkeys.

A brain-tumor model in adult monkeys may be significant because of the biological similarity to humans as well as the feasibility for surgical manipulation and for sequential computerized tomography (CT) scanning. In the present study, brain tumors were successfully produced in Japanese monkeys (Macaca fuscata), each weighing 2 to 10.8 kg, with an average age of 5.1 years old. Tumor cells were implanted by intracerebral inoculation of 4 X 10(7) chick embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV). With a 15- to 67-day latency, brain tumors were induced in 11 (73.3%) of 15 RSV-inoculated monkeys. Contrast-enhanced CT scans delineated all solitary intracerebral tumors greater than 4 to 6 mm in diameter. The CT images were proved at autopsy to be accurate within 2 mm in determining the size of tumor. Five of the 11 monkeys with intracerebral tumors died, with an average survival time of 26.6 days after RSV inoculation. The induced tumors were classified as either glioma or sarcoma by the presence or absence of glial fibrillary acidic protein (GFAP) and S-100 protein. A chromosome analysis of cultured tumor cells showed a diploid number of 42, indicating monkey origin. It is concluded that the reproducible brain tumor in the adult Japanese monkey inoculated with RSV can serve as a good experimental brain-tumor model for the further study of human malignant brain tumors.

Immunohistochemical demonstration of DNA polymerase alpha in human brain-tumor cells.

The proliferative capacity of brain-tumor cells was analyzed in vitro and in situ using monoclonal antibody (MAb) against deoxyribonucleic acid (DNA) polymerase alpha. For the in vitro studies, two cultured human glioma cell lines were investigated using MAb against DNA polymerase alpha, the MAb Ki-67, a serum against proliferating cell nuclear antigen (PCNA/cyclin), bromodeoxyuridine (BUdR), and an anti-BUdR MAb. During exponential growth of the cells, the percentage of polymerase alpha-positive cells (the "polymerase alpha score") ranged from 72.0% to 77.1%, the Ki-67-positive cells (the "Ki-67 score") ranged from 43.4% to 59.4%, the PCNA/cyclin-positive cells from 30.9% to 41.4%, and the BUdR labeling index from 28.6% to 39.3%. For the in situ studies, tissue from 60 human brain tumors and from two normal human brains was investigated and the polymerase alpha scores and Ki-67 scores were compared. In normal brain tissue, no immunostaining was found by either method. In brain tumors, both the polymerase alpha scores and the Ki-67 scores correlated with the histological grade of malignancy. Polymerase alpha scores were generally higher than Ki-67 scores in the same specimen, especially in malignant brain tumors. These findings suggest that immunostaining of DNA polymerase alpha is a convenient and important new method by which to estimate the cellular proliferation rate of brain tumors. Polymerase alpha scores may be closer to the growth fraction of the individual tumor than the MAb Ki-67 or other scores.

Effects of hyperbaric oxygenation on cerebral vasomotor tone in acute intracranial hypertension: an experimental study.

The effects of hyperbaric oxygenation with 2 atm. pressure and 100 percent oxygen on cerebrovascular tone were assessed by the reactivity of the cerebral vessels to CO2 and vasomotor capacitance index (the cranial pressure divided by the mean arterial pressure) in 50 anaesthetized artifically ventilated dogs, in which intracranial pressure was raised by slow inflation of an extradural balloon. Hyperbaric oxygenation reduced the intracranial pressure only at the stage when the cerebral vessels were still responsive to CO2, as indicated by a rise in intracranial pressure of 30-70 mmHg; under these circumstances, both the reactivity of CO2 and the vasoconstrictor tone of cerebral vessels were improved by hyperbaric oxygenation. At this stage, rapid decrease of the intracranial pressure produced by deflation of the extradual balloon showed no rebound, and hyperbaric oxygenation rapidly restored the vasomotor tone. When CO2 failed to influence the intracranial pressure of about 100 mmHg hyperbaric oxygenation did not aid the recovery of the reactivity of the cerebral vessels to CO2 or the vasoconstrictor tone. In extreme intracranial hypertension (above 100 mmHg) when there was reactivity to CO2 and the electroencephalogram was flat, rapid balloon deflation was followed by a further gradual increase of intracranial pressure and hyperbaric oxygenation did not restore the cerebrovascular tone. The effect of hyperbaric oxygenation in experimental intracranial hypertension appeared to be dependent upon the vasoconstrictor tone of the cerebral vessels, which would be indicated by a vasodilator response to CO2.

Increase of peripheral natural killer T cells in hemodialysis patients.

AIMS: Patients with end-stage renal disease often present a state of immunodeficiency. To elucidate the involvement of natural killer T (NKT) cells in the development of pathologies associated with uremia, we examined the percentages and characteristics of NKT cells (CD56+ T cells and CD57+ T cells) in peripheral lymphocites from hemodialysis (HD) patients. MATERIALS AND METHODS: Thirty-three patients who had undergone HD therapy for 2 to 5 years (group A, n = 15) or for more than 10 years (group B, n = 18) and 17 normal controls participated in this study. Cell surface antigens of lymphocytes were analyzed using immunofluorescent flow cytometry. RESULTS: The percentage of peripheral CD56+ T cells was increased in group A and B compared with controls, and that of CD57+ T cells was increased in group B compared with controls and group A. The most abundant population in CD56+ T cells was DN T cells, and that in CD57+ T cells was CD8+ T cells. In CD57+ T cells, a higher proportion of CD8+ T cells and a lower proportion of DN T cells were found in group B compared with controls. CONCLUSIONS: We detected increases of CD56+ T cells and CD57+ T cells in HD patients. The elevation of NKT cells might affect some complications associated with HD.