Chimera Time-Crystalline Order in Quantum Spin Networks.

Symmetries are well known to have had a profound role in our understanding of nature and are a critical design concept for the realization of advanced technologies. In fact, many symmetry-broken states associated with different phases of matter appear in a variety of quantum technology applications. Such symmetries are normally broken in spatial dimension, however, they can also be broken temporally leading to the concept of discrete time symmetries and their associated crystals. Discrete time crystals (DTCs) are a novel state of matter emerging in periodically driven quantum systems. Typically, they have been investigated assuming individual control operations with uniform rotation errors across the entire system. In this work we explore a new paradigm arising from nonuniform rotation errors, where two dramatically different phases of matter coexist in well defined regions of space. We consider a quantum spin network possessing long-range interactions where different driving operations act on different regions of that network. What results from its inherent symmetries is a system where one region is a DTC, while the second is ferromagnetic. We envision our work to open a new avenue of research on chimeralike phases of matter where two different phases coexist in space.

p.E66Q mutation in the GLA gene is associated with a high risk of cerebral small-vessel occlusion in elderly Japanese males.

BACKGROUND AND PURPOSE: GLA is the causative gene of Fabry disease, an X-linked lysosomal storage disorder resulting from α-galactosidase A (α-GAL) deficiency. Stroke is an important manifestation of Fabry disease, and recent epidemiological studies have indicated that up to 4.9% of young male cryptogenic stroke patients have GLA mutations. To determine the importance of GLA mutations in the general stroke population, the frequency of GLA mutations in Japanese male ischaemic stroke (IS) patients with various risk factors and ages was measured. METHODS: A total of 475 male IS patients (mean age 69.7 ± 12.5 years), were enrolled in this study. A blood sample was obtained to produce blood spots for measurement of α-GAL activity. Blood samples with decreased enzymatic activity were reassayed and the entire GLA gene was analyzed by direct DNA sequencing if α-Gal A activity was consistently low. RESULTS: α-Gal A activity was decreased in 10 men, five of whom (1.1%) had the GLA gene mutation, p.E66Q. All IS patients with p.E66Q mutation had substantial residual α-Gal A activity, in contrast to patients with classic-type Fabry disease. Clinically, all patients with p.E66Q mutation were > 50 years old and had multiple small-vessel occlusions (lacunar infarctions). Statistical analysis using Fisher's exact test showed the allele frequency of GLA p.E66Q in patients with small-vessel occlusion to be significantly higher than that in the general Japanese population [odds ratio (OR) = 3.34, P = 0.025). CONCLUSIONS: GLA p.E66Q mutation is a genetic risk factor for cerebral small-vessel occlusion in elderly Japanese males.

[Delayed augmentation effect of cytokine production after hyperthermia stimuli].

Heatstroke is considered an important condition that may contribute to endothelial cell damage. The aim of this study was to assess temporal profiles of the cytokine (IL-6 and IL-8) and mRNA production when endothelial cells undergo higher temperature stimuli. In the first group, human umbilical vascular endothelial cells (HUVECs)were cultured at 4 different temperatures (37, 38, 39 or 40 degrees C) for 1, 3 and 5 h. In the second group, HUVECs were cultured at 37 degrees C for 4 h or 23 h, after stimulation by heating for one hour at the same culture temperatures used in the first group (37 degrees C to 40 degrees C). After culturing, IL-6 and IL-8 mRNA and protein levels were measured. It has been found the cytokine mRNA levels being significantly higher (p < 0.001) in all cells incubated at higher temperatures than those in the control (cultivation at 37 degrees C). At the same time, the productionof IL-6 and 8 at a higher temperature (39, 40 degrees C) was significantly lower (p < 0.001) than at 37 degrees C (control), and the decrease was temperature dependent. However, IL-6 and IL-8 levels were significantly greater in the cells at 23 h after transient hyperthermic (40 degrees C, 1 h) stimulation than in control ones (p < 0.001).After a transient hyperthermia, the production of the cytokines in HUVECs is initially inhibited and then augmented. The results indicated that tissue injury might continue to develop after a hyperthermic event. There might be a potent risk for underestimation of cytokine induced tissue injury in the acute phase of a heatstroke.

Exercise effects on methylation of ASC gene.

Chronic moderate exercise has been reported to reduce pro-inflammatory cytokines. To analyze the molecular mechanisms by which training exerts these effects, the epigenetic influences of age and exercise on the ASC gene, which is responsible for IL-1beta and IL-18 secretion, were investigated by ASC gene methylation. Further, the relationship between carcinogenesis and exercise, and methylation of the P15 tumor suppressive gene was also analyzed. High-intensity interval walking exercise, consisting of 3 min low-intensity walking at 40% of peak aerobic capacity followed by a 3 min high-intensity walking period above 70% of peak aerobic capacity, was continued for 6 months. Peripheral blood DNA extracts from young control (n=34), older control (n=153), and older exercise (n=230) groups were then analyzed by pyrosequencing for DNA methylation. Methylation of ASC decreased significantly with age (young control vs. older control, p<0.01), which is indicative of an age-dependent increase in ASC expression. Compared to the older control group, the degree of ASC methylation was higher in the older exercise group (older control vs. older exercise: p<0.01), and presumably lower ASC expression. Neither exercise nor age affected the methylation of the P15. In summary, chronic moderate exercise appears to attenuate the age-dependent decrease in ASC methylation, implying suppression of excess pro-inflammatory cytokines through reduction of ASC expression.

A clinical investigation of the association between perioperative oral management and prognostic nutritional index in patients with digestive and urinary cancers.

Background: The prognostic nutritional index (pni) is a simple metric calculated using serum albumin and the peripheral lymphocyte count. It was reported that a low pni score is significantly associated with major postoperative complications and poor prognosis. The purpose of the present study was to investigate the effects of perioperative oral management (pom) on the perioperative pni profiles of patients with digestive system or urinary cancers. Study Design: The medical records of 181 patients with cancer who underwent surgery and for whom a pni could be calculated were retrospectively reviewed. Results: The intervention rate with pom was 34.8%. The median preoperative pni score was 48.25 in all patients with a pom intervention [25% to 75% interquartile range (iqr): 44.38-54.13] and 47.25 in those without an intervention (iqr: 42.0-53.5). Compared with patients not receiving pom, those who received pom had significantly higher pni scores from the early postoperative period (p < 0.05). Notably, of patients who could resume oral intake within 3 days after surgery, those who received pom intervention, compared with those who did not, had significantly higher pni scores from the early postoperative period (p < 0.05). Conclusions: Perioperative oral management interventions might have positive effects on the postoperative pni scores of patients with cancer.

Whole-genome sequencing of Staphylococcus epidermidis bloodstream isolates from a prospective clinical trial reveals that complicated bacteraemia is caused by a limited number of closely related sequence types.

OBJECTIVES: The significance of isolating Staphylococus epidermidis from a blood culture is highly heterogeneous, ranging from contamination to an indication of a serious infection. Herein we sought to determine whether there is a relationship between S. epidermidis genotype and clinical severity of bacteraemia. METHODS: S. epidermidis bacteraemias from a prospective, multicentre trial at 15 centres in the United States and one in Spain were classified as simple (including possible contamination), uncomplicated, and complicated. Whole-genome sequencing (WGS) was performed on 161 S. epidermidis isolates, and clinical outcomes were correlated with genotypic information. RESULTS: A total of 49 S. epidermidis sequence types (STs) were identified. Although strains of all 49 STs were isolated from patients with either simple or uncomplicated infection, all strains causing complicated infections were derived from five STs: ST2, ST5, ST7, ST16, and ST32. ST2 and ST5 isolates were significantly more likely to cause uncomplicated and complicated bloodstream infections compared to simple bacteraemia (odds ratio 2.0, 95%CI 1.1-3.9, p 0.04). By multivariate regression analysis, having an ST2 or ST5 S. epidermidis bacteraemia was an independent predictor of complicated bloodstream infection (odds ratio 3.7, 95%CI 1.2-11.0, p 0.02). ST2/ST5 strains carried larger numbers of antimicrobial resistance determinants compared to non-ST2/ST5 isolates (6.34 ± 1.5 versus 4.4 ± 2.5, p < 0.001). CONCLUSION: S. epidermidis bacteraemia was caused by a genetically heterogeneous group of organisms, but only a limited number of STs-particularly multidrug-resistant ST2 and ST5 strains-caused complicated infections.

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation.

AIMS: To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called 'ion beam'. METHODS AND RESULTS: Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8-azaadenine- and 28 classes of 8-azaaguanine-resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8-azaadenine-resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8-azaaguanine-resistant mutants, it was shown that mutant no. G81-3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3.1 and 1.8 g l(-1) on 21-day culture for mutant no. G81-3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. CONCLUSIONS: The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large-scale production of cordycepin.

Screening of nineteen unrelated families with generalized resistance to thyroid hormone for known point mutations in the thyroid hormone receptor beta gene and the detection of a new mutation.

Generalized resistance to thyroid hormone (GRTH) is a syndrome characterized by impaired tissue responsiveness to thyroid hormone. Two distinct point mutations in the hormone binding domain of the thyroid hormone receptor (TR) beta have recently been identified in two unrelated families with GRTH. One, Mf, involves a replacement of the normal glycine-345 for arginine in exon 7 and another, Mh, replaces the normal proline-453 for histidine in exon 8. To probe for the presence of the Mf and Mh defect in 19 unrelated families with GRTH, we applied separate polymerase chain reactions using allele-specific oligonucleotide primers containing the normal and each of the two mutant nucleotides at the 3'-position. A total of 24 affected subjects and 13 normal family members were studied. The mode of inheritance was dominant in 13 families, was unknown in 5 families, and was clearly recessive in 1 family in which only the consanguineous subjects were affected. Primers containing the substitutions specific for Mf and Mh amplified exons 7 and 8, respectively, only in affected members of each of the two index families. Primers containing the normal sequences amplified exons 7 and 8 of the TR beta gene in all subjects except affected members of one family. In this family with recessively inherited GRTH, neither exon could be amplified using any combinations of primers and DNA blot revealed absence of all coding exons. These results indicate a major deletion of the TR beta gene, including both DNA and hormone binding domains. Since heterozygous members of this family are not affected, the presence of a single normal allele is sufficient for normal function of the TR beta. These data also support the hypothesis that in the dominant mode of GRTH inheritance the presence of an abnormal TR beta interferes with the function of the normal TR beta. Distinct mutations are probably responsible for GRTH in unrelated families.

Organ dysfunction assessment score for severe head injury patients during brain hypothermia.

The purpose of this study was to evaluate the utility of a novel organ dysfunction assessment score developed for patients with severe traumatic brain injury during therapeutic brain hypothermia. The Brain Hypothermia Organ Dysfunction Assessment (BHODA) score is calculated through the combined assessment of 6 indices: central nervous system (CNS) function, respiratory function, cardiovascular function, hepatosplanchnic circulation, coagulation, and metabolism. The CNS, hepatosplanchnic circulation, and metabolic indices were based on measurements of cerebral perfusion pressure, gastric tonometry, and blood glucose, respectively. Thirty-nine patients with severe closed head injuries (scores of 3 to 8 on the Glasgow Coma Scale) were enrolled. Seven patients (18%) died during hospitalization. Outcome was favorable in 20 patients and unfavorable in 19. The BHODA score proved useful in describing sequences of complications during therapeutic brain hypothermia. A total maximum BHODA score of more than 13 points corresponded to a mortality of 70%. In a multivariate model, the total maximum BHODA score was independently associated with neurological outcome (odds ratio for unfavorable neurological outcome, 2.590: 95% confidence interval, 1.260, 5.327). In conclusion, the BHODA score can help assess multiple organ dysfunction/failure during therapeutic hypothermia and may be useful for predicting outcome.

Rewarming following accidental hypothermia in patients with acute subdural hematoma: case report.

A 57-year-old man was admitted to the Emergency and Critical Care Department with accidental hypothermia (31.5 degrees C) after resuscitation from cardiopulmonary arrest (CPA). Brain CT revealed an acute subdural hematoma. Active core rewarming to 33 degrees C was performed using an intravenous infusion of warm crystalloid. The patient underwent craniotomy soon after admission, with bladder temperature maintained at 33 to 34 degrees C throughout the surgery. Therapeutic hypothermia (34 degrees C) was continued for 2 days, followed by gradual rewarming. After rehabilitation, the patient was able to continue daily life with assistance. Traumatic brain injury (TBI) following CPA is associated with extremely unfavorable outcomes. Very few patients with acute subdural hematomas presenting with accidental hypothermia and CPA have been reported to recover. No suitable strategies have been clearly established for the rewarming performed following accidental hypothermia in patients with TBI. Our experience with this patient suggests that therapeutic hypothermia might improve the outcome in some patients with severe brain injury. It also appears that the method used for rewarming might play an important role in the therapy for TBI with accidental hypothermia.

Acceleration of chemokine production from endothelial cells in response to lipopolysaccharide in hyperglycemic condition.

UNLABELLED: Chronic hyperglycemia is an established risk factor for endothelial damage. It remains unclear, however, whether brief hyperglycemic episodes after acute stress alter the function of vascular endothelial cells in response to endotoxin. We hypothesize that brief hyperglycemic episodes enhance the production of interleukin-8 (IL-8) after lipopolysaccharide (LPS) stimulation. METHODS: Human umbilical vein endothelial cells (HUVECs; 1 x 10(5) cells/mL, cells from subcultures 2-5, n = 6) were cultivated in various concentrations of glucose (200, 300, 400, and 500 mg/dL) with or without LPS stimulation (1 microg/mL) for 24 hours. After culture, IL-8 levels in the supernatant were measured using ELISA. RESULTS: HUVECs cultured at glucose concentrations of 300 and 400 mg/dL produced more (p < 0.01) IL-8 than control cells (200 mg/dL). HUVECs cultured at glucose concentrations of 300 and 400 mg/dL also produced more (p < 0.01) IL-8 than those cultured in the absence of LPS. CONCLUSIONS: Hyperglycemic conditions enhance IL-8 production by vascular endothelial cells, and this response is augmented by LPS. Infections may foster neutrophil accumulation at injury sites. These results suggest that it is important to manage even short-term increases in blood glucose after acute stress.

Importance of cerebral perfusion pressure management using cerebrospinal drainage in severe traumatic brain injury.

OBJECTIVE: To evaluate hemodynamics in patients with severe traumatic brain injury (TBI) after cerebral perfusion pressure (CPP) management using cerebrospinal fluid (CSF) drainage. METHODS: Twenty-six patients with TBI (Glasgow Coma Score = 8 or less) were investigated. Mean arterial blood pressure, CPP, cardiac index (CI), systemic vascular resistance index (SVRI), and central venous pressure were measured. The patients were divided into 2 groups after craniotomy: the intraparenchymal ICP (IP-ICP) monitoring group (n = 14) and ventricular ICP (V-ICP) monitoring group (n = 12). Patient hemodynamics were investigated on the second hospital day to identify differences. Measurements indicated a target CPP above 70 mmHg and a central venous pressure of 8 10 mmHg in both groups. Mannitol administration (IP-ICP group) or CSF drainage (V-ICP group) was performed whenever the CPP remained below 70 mmHg. RESULTS: High SVRI and low CI (p < 0.05) were observed in the IP-ICP group. The V-ICP group exhibited a reduction in the total fluid infusion volume of crystalloid (p < 0.01) and a reduction in the frequency of hypotensive episodes after the mannitol infusion. CONCLUSIONS: CPP management using CSF drainage decreases the total infusion volume of crystalloid and may reduce the risk of aggravated brain edema after excess fluid resuscitation.

Clinical characteristics of postoperative contralateral intracranial hematoma after traumatic brain injury.

OBJECTIVES: To investigate the clinical characteristics of contralateral intracranial hematoma (ICH) after traumatic brain injury. METHODS: The subjects included 149 patients with traumatic ICH treated by hematoma evacuation. The patients were retrospectively divided into a bilateral ICH (B-ICH) group and unilateral ICH (U-ICH) group after craniotomy using brain CT scans for comparison of the following parameters: complicated expanded brain bulk from the cranial window, hypotension during craniotomy, and outcome. RESULTS: Post-craniotomy brain CT scans revealed U-ICH in 106 patients and B-ICH in 43 patients. Average Glasgow Coma Scale on arrival did not differ between the groups, but a higher proportion of patients in the B-ICH group deteriorated after admission (p = 0.02). The B-ICH patients also exhibited a significantly higher rate of expanded brain bulk from the cranial window (p < 0.05). No significant difference was observed between the groups with hypotension during craniotomy. The B-ICH group exhibited a lower rate of favorable outcome (p < 0.05) and higher mortality (p < 0.05). CONCLUSION: The B-ICH patients had a worse outcome than the U-ICH patients. Contralateral ICH was difficult to forecast based on pre- and intraoperative clinical conditions. Subdural hematoma or contusional ICH was frequently observed as a contralateral ICH.

Changes in coagulative and fibrinolytic activities in patients with intracranial hemorrhage.

OBJECTIVE: To investigate whether any changes occur in the coagulative/fibrinolytic cascade in patients with subarachnoid hemorrhage (SAH) or hypertensive intracerebral hemorrhage (HICH). DESIGN AND METHODS: Subjects included 143 patients with intracranial hemorrhage (SAH, n = 50; HICH, n = 82; ROSC-SAH [return of spontaneous circulation after cardiopulmonary arrest due to SAH], n = 11). Coagulative and fibrinolytic factors were measured in blood samples taken on admission. RESULTS: The prothrombin fragment 1+2 level was significantly higher (p < 0.005) in SAH patients than in HICH patients. The fibrinolytic factors (plasmin alpha 2-plasmin inhibitor complex, D-dimer, or fibrinogen degradation products) in SAH and ROSC-SAH were both significantly higher than those in HICH, but the significance of difference was stronger in the case of ROSC-SAH (p < 0.05). DISCUSSION: Both coagulative and fibrinolytic activities were altered after the onset of SAH. These results demonstrate that the coagulative/fibrinolytic cascade might be activated via different mechanisms in different types of stroke. It remains unclear, however, whether a significant alteration of the fibrinolytic cascade in patients with ROSC-SAH might be a nonspecific phenomenon attributable to the reperfusion after collapse.

Brain oxygen metabolism may relate to the temperature gradient between the jugular vein and pulmonary artery after cardiopulmonary resuscitation.

OBJECTIVE: A gradient between the jugular vein temperature and core body temperature has been reported in animal and clinical studies; however, the pathophysiological meaning of this phenomenon remains unclear. This study was conducted to identify the temperature gradient between the jugular vein and pulmonary artery in comatose patients after cardiopulmonary resuscitation. MATERIALS AND METHODS: The temperatures of the jugular vein and pulmonary artery were measured in 19 patients at 6 and 24 hours after cardiopulmonary resuscitation. Jugular venous blood saturation (SjO2; %) was also measured concomitantly. The patients were divided into 2 groups: high SjO2 (SjO2 > 75%: H-group; n = 10) and normal SjO2 (SjO2 < or = 75%: N-group; n = 9). The temperature gradient was calculated by subtracting the temperature of the pulmonary artery from that of the jugular vein (jugular - pulmonary = dT degrees C). Statistical significance was defined as p < 0.05. RESULTS: dT was significantly lower in the H-group than in the N-group at 6 hours (0.120 +/- 0.011: mean +/- SD vs. 0.389 +/- 0.036: p = 0.0012) and 24 hours (0.090 +/- 0.005 vs. 0.256 +/- 0.030: p = 0.0136) after cardiopulmonary resuscitation. CONCLUSION: The temperature gradient between the jugular vein and pulmonary artery was significantly lower in patients with high SjO2 after cardiopulmonary resuscitation. This temperature gradient may be reflected in brain oxygen metabolism.

Relationship of hypoglycemia to tumor necrosis factor production and antitumor activity: role of glucose, insulin, and macrophages.

The role of hypoglycemia in tumor necrosis factor (TNF) production was examined. TNF was produced from sera of animals presensitized with reticuloendothelial system stimulants after lipopolysaccharide (LPS) challenge. Blood glucose was strongly reduced during TNF production. Glucose administration to presensitized mice (before LPS challenge) caused inhibition of TNF production. Exogenous insulin injection inhibited TNF production in a dose-related manner. Peritoneal exudate cells (PEC) from Propionibacterium acnes-primed mice revealed increased glucose consumption during in vitro TNF production but showed no relationship between the degree of glucose consumption and the ability to produce TNF. Insulin addition to the culture medium caused inhibition of TNF production from PEC, which indicated that insulin may block TNF production from macrophages. Administration of highly purified TNF (without concomitant LPS) induced extensive tumor necrosis but did not induce hypoglycemia; LPS induced moderate necrosis with accompanying hypoglycemia; insulin induced hypoglycemia but did not induce tumor necrosis. It is concluded that hypoglycemia does not accompany the action of TNF.

Differanisole A, a novel antitumor antibiotic, enhances growth inhibition and differentiation of human myeloid leukemia cells induced by 9-cis retinoic acid.

Differanisole A, 3,5-dichloro-2-hydroxy-4-methoxy-6-n-propylbenzoic acid, inhibited growth of human myeloid leukemia cells. The compound induced G1 arrest and granulocytic differentiation of HL-60 cells, although the differentiation-inducing effect was modest. Differanisole A and 9-cis retinoic acid (9cisRA) synergistically inhibited the growth and induced functional and morphologic differentiation of HL-60 and NB4 cells, whereas the combined treatment with differanisole A and all-trans retinoic acid or 1alpha,25-dihydroxyvitamin D3 was less effective. Similar results were obtained in primary culture of leukemia cells from a patient with acute promyelocytic leukemia. The synergistic effect on growth inhibition and induction of differentiation required simultaneous treatment with differanisole A and 9cisRA. Differanisole A and an RXR-specific ligand (Ro47-5944) cooperatively inhibited the cell growth, while the combined effect of differanisole A and an RAR-specific ligand Am80 was just additive. Differanisole A in combination with 9cisRA may have implications for therapy of acute promyelocytic leukemia patients.

Phytochrome, Gibberellins, and Hypocotyl Growth (A Study Using the Cucumber (Cucumis sativus L.) long hypocotyl Mutant).

The possible involvement of gibberellins (GAs) in the regulation of hypocotyl elongation by phytochrome was examined. Under white light the tall long hypocotyl (lh) cucumber (Cucumis sativus L.) mutant, deficient in a type B-like phytochrome, shows an increased "responsiveness" (defined as response capability) to applied GA4 (the main endogenous active GA) compared to the wild type. Supplementing far-red irradiation results in a similar increase in responsiveness in the wild type. Experiments involving application of the precursor GA9 and of an inhibitor of GA4 inactivation suggest that both the GA4 activation and inactivation steps are phytochrome independent. Endogenous GA levels of whole seedlings were analyzed by combined gas chromatography-mass spectrometry using deuterated internal standards. The levels of GA4 (and those of GA34, the inactivated GA4) were lower in the lh mutant under low-irradiance fluorescent light compared with the wild type, similar to wild type under higher irradiance light during the initial hypocotyl extension phase, and higher during the phase of sustained growth, in which extension involved an increase in the number of cells in the upper region. In all cases, growth of the lh mutant was more rapid than that of the wild type. It is proposed that GA4 and phytochrome control cell elongation primarily through separate mechanisms that interact at a step close to the terminal response.

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin.

ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.

Expression of three forms of thyroid hormone receptor in human tissues.

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.