Juzentaihoto extract suppresses adipocyte hypertrophy and improves hyperglycemia in KKAy mice.

Juzentaihoto is a herbal medicine with reported anti-inflammatory effects, and it is predicted to improve inflammation and insulin sensitivity within obesity. In the present study, juzentaihoto hot water extract (JTT) was administered to obese type 2 diabetic model mice (KKAy) for 56 days. In addition, the effects of JTT on the adipose tissue, glucose metabolism, and blood lipids were evaluated for examining its impact on insulin sensitivity and obesity. As a result of JTT administration, KKAy mice exhibited suppressed adipocyte hypertrophy, decreased the mRNA levels of tumor necrosis factor α, and increased the mRNA levels of adiponectin in epididymal fat tissue. In addition, fasting blood glucose levels, blood triglyceride, and total cholesterol decreased. In summary, these data indicated that JTT administration suppressed the production of inflammatory cytokines and increased adiponectin levels in the adipose tissue. Therefore, with improved insulin sensitivity, blood glucose, and lipid decreased.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Generation of continuous large granular lymphocyte lines by interleukin 2 from the spleen cells of mice infected with Moloney leukemia virus. Involvement of interleukin 3.

Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.

Interleukin 7 production and function in stromal cell-dependent B cell development.

The role of IL-7 in the stromal cell-dependent B cell development was investigated using two stromal cell clones, ST2 and PA6; the former supports B lymphopoiesis while the latter can not. We demonstrate here that: (a) the ability of the stromal cell clone to produce IL-7 correlates well with the stromal cell activity to support B lymphopoiesis; (b) IL-7 production by ST2 is inducible rather than constitutive; (c) the IL-7-dependent B cell itself is a potent inducer of IL-7 production by ST2; (d) addition of rIL-7 to the PA6 layer renders this in vitro environment B lymphopoietic; and (e) the differentiation from early B progenitor to pre-B cell requires both IL-7 and other stromal cell molecule(s) yet to be identified.

A recombinant murine granulocyte/macrophage (GM) colony-stimulating factor derived from an inducer T cell line (IH5.5). Functional restriction to GM progenitor cells.

The cDNA for the murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was cloned from a cDNA library obtained from a murine T cell line, IH5.5, by using two synthetic probes that encoded two parts of the GM-CSF from murine lung. The cDNA inserted into the plasmid vector pcDV1 was transfected into monkey COS-1 cells and the conditioned medium was used to investigate the hemopoietic activities of the resultant product, recombinant GM-CSF (rGM-CSF), by means of various colony assays. rGM-CSF stimulated only neutrophil/macrophage colonies in the cultures of murine normal bone marrow and fetal liver cells. No other colony stimulating activities (CSA) were seen in the preparation including burst-promoting activity, eosinophil-CSA, megakaryocyte-CSA and mast cell-CSA. rGM-CSF could not support colony formation of 5-fluorouracil-treated mouse spleen cells, in which only the primitive population of stem cells survived. However, after culture of these cells with PWM-spleen cell-conditioned medium (PWM-SCM), the colonies consisting of blast cells were formed. These blast cells could now be induced to form neutrophil/macrophage colonies in the presence of rGM-CSF. Pure neutrophil colonies, pure macrophage colonies, as well as mixed neutrophil/macrophage colonies, were formed from these single blast cells in the presence of rGM-CSF by micromanipulation. rGM-CSF did not act on pluripotent hemopoietic stem cells, but did act directly and selectively on neutrophil/macrophage progenitors. Moreover, striking heterogeneities were noted in the size of the colonies and the proportion of components. GM-CSF is, therefore, considered to play a noninstructive role in the differentiation of the GM pathway.

New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome.

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes.

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

Joining of the c-myc gene and a line 1 family member on chromosome 8 in a human primary giant cell carcinoma of the lung.

A rearranged c-myc gene found in a human primary giant cell carcinoma of the lung was analyzed. The rearrangement was found in the region about 6 kilobase pairs upstream of the c-myc gene. The breakpoint was joined to a sequence carrying a Line 1 (L1) family member located on chromosome 8. This in vivo rearrangement of the c-myc gene specific to tumor cells may represent one mechanism of activation of a protooncogene during tumorigenesis or tumor progression in human cancer.

Loss of the same alleles of HRAS1 and D11S151 in two independent pancreatic cancers from a patient with multiple endocrine neoplasia type 1.

The DNAs from two independent pancreatic cancers (tumors 1 and 2) in a patient with multiple endocrine neoplasia type 1 were analyzed. No amplification or gross rearrangement of 19 protooncogenes was observed. However, Southern blot analysis using polymorphic DNA probes revealed loss of heterozygosity at loci on chromosome 11p in both tumors. In tumor 1, an extensive region including the HRAS1, PTH, CALCA, and D11S151 loci was deleted, while in tumor 2 loss of heterozygosity was limited at the HRAS1 and D11S151 loci. Because loss of heterozygosity at other chromosomal loci in the two tumors was quite rare, loss of genes on 11p might be nonrandom. It is noteworthy that the same allele at the HRAS1 locus and also the same allele at the D11S151 locus were lost in the two independent tumors. These results suggest that loss of genes at the HRAS1 and/or D11S151 loci plays an important role unmasking the remaining sequences probably having a recessive mutation.

Loss of heterozygosity at 11p15 in malignant glioma.

Deletions of loci on chromosome 11p have been found frequently in several malignant tumors including gliomas, suggesting the presence of tumor suppressor genes. We analyzed 38 gliomas [26 malignant gliomas (grades III and IV) and 12 less malignant gliomas (grade I and II)] for loss of heterozygosity using microsatellite sequences on 11p as polymorphic markers. Loss of heterozygosity was found in 8 of 26 malignant gliomas (31%) but not in the less malignant gliomas. In the region with loss of heterozygosity, loci on 11p15.5-pter were commonly deleted. Our results suggest that a putative tumor suppressor gene involved in malignant progression of gliomas is located in an approximately 21-cM region on 11p15.5-pter.

Distribution of respiration-related neuronal activity in the thoracic spinal cord of the neonatal rat: An optical imaging study.

The inspiratory motor outputs are larger in the intercostal muscles positioned at more rostral segments. To obtain further insights into the involvement of the spinal interneurons in the generation of this rostrocaudal gradient, the respiratory-related neuronal activities were optically recorded from various thoracic segments in brainstem-spinal cord preparations from 0- to 2-day-old rats. The preparation was stained with a voltage-sensitive dye, and the optical signals from about 2.5s before to about 7.7s after the peak of the C4 inspiratory discharge were obtained. Respiratory-related depolarizing signals were detectable from the ventral surface of all thoracic segments. Since the local blockage of the synaptic transmission in the thoracic spinal cord induced by the low-Ca(2+) superfusate blocked all respiratory signals, it is likely that these signals came from spinal neurons. Under the-low Ca(2+) superfusate, ventral root stimulation, inducing antidromic activation of motoneurons, evoked depolarizing optical signals in a restricted middle area between the lateral edge and midline of the spinal cord. These areas were referred to as 'motoneuron areas'. The respiratory signals were observed not only in the motoneuron areas but also in areas medial to the motoneuron areas, where interneurons should exist; these were referred to as 'interneuron areas'. The upper thoracic segments showed significantly larger inspiratory-related signals than the lower thoracic segments in both the motoneuron and interneuron areas. These results suggest that the inspiratory interneurons in the thoracic spinal cord play a role in the generation of the rostrocaudal gradient in the inspiratory intercostal muscle activity.

A model to estimate the increase of genetic variability due to electrophoretically cryptic alleles.

To consider the problem of the increase of genetic variability due to electrophoretically cryptic alleles, the equally degenerate electromorph model is proposed. Mutation, random sampling drift and selection can be incorporated in this model. Simple formulas are obtained to show how genetic variability increases when cryptic alleles are distinguished. This model is extended to a two-population system to see the effect of cryptic allele variation on genetic differentiation.

Persistence of repeated sequences that evolve by replication slippage.

The evolution of short repeated sequences by replication slippage under the assumption of selective neutrality is modeled using a linear birth and death process. The equilibrium distribution, the distribution of the life expectancy of a repeated sequence when the process starts from two repeats, the age distribution of repeats, the probability of obtaining two genes with i and j copies which diverged t generations ago and the conditional variance of copy number given the repeat number is more than one are computed. The distributions of life expectancy and age are shown to have long tails. Also the statistic which estimates the conditional variance is shown to have a large coefficient of variation. Using these theoretical results, we develop an approximate test of our model and analyze persistent repeated sequences found in the primate beta-globin gene region and Oenothera chloroplast DNA which are polymorphic within species. We found one sequence in Oenothera chloroplast DNA which does not fit to our neutral model.

A population genetic study of the evolution of SINEs. I. Polymorphism with regard to the presence or absence of an element.

SINEs are short interspersed repeated DNA elements which are considered to spread throughout genomes via RNA intermediates. Polymorphisms with regard to the presence or absence of SINE are occasionally observed in a specific location of a genome. We modeled the evolution of SINEs with regard to this type of polymorphism. Because SINEs are rarely deleted, multiplication of elements is confined to a certain period, and a few master copies are considered to be responsible for their multiplication, the usual population genetic models of transposable elements assuming the equilibrium state are not applicable to describe the evolution of SINEs. Taking into account these properties and assuming selective neutrality, we computed conditional probabilities of finding a SINE at a specific site given that this site is first found because it is occupied by a SINE in an original sample. Using these probabilities, we investigated ways to estimate the multiplication period and infer relationships among populations. The latter inference procedures are shown to be strongly dependent on the multiplication period.

Estimation of left ventricular elastance without altering preload or afterload in the conscious dog.

OBJECTIVE: The aim was to determine the slope (EES) of the left ventricular end systolic pressure-volume line (ESPVL) without altering preload or afterload in conscious dogs. METHODS: Dogs (n = 10) were instrumented to determine left ventricular volume from ultrasonic left ventricular internal dimensions, and to measure left ventricular pressure using a micromanometer. Studies were performed one to two weeks after instrumentation while the animals were conscious. ESPVL was determined from variably loaded left ventricular pressure-volume (P-V) loops generated by the vena caval occlusion. Contractile state was increased by intravenous dobutamine (8 micrograms.kg-1 x min-1) and decreased by intravenous verapamil (10 mg) given after autonomic blockade. From a single normally ejecting beat, we calculated EES-single beat (mm Hg.ml-1) as peak isovolumetric pressure (Pmax) minus end systolic pressure divided by stroke volume. Sunagawa's technique was used to estimate Pmax by fitting the pressure during the isovolumetric contraction and relaxation as: P(t) = 1/2 X Piso[1-cos(omega t+c)]+LVEDP, where Piso = peak isovolumetric developed pressure, LVEDP = left ventricular end diastolic pressure, c = constant accounting for variations in phase angle, and omega = 2 pi/T in which T is duration of contraction. RESULTS: After dobutamine, EES increased, from 8.9(SEM 0.8) to 12.5(1.0) mm Hg.ml-1 (p < 0.05), and EES-single beat increased from 9.1(0.9) to 12.0(1.4) mm Hg.ml-1 (p < 0.05). Conversely, after verapamil, EES decreased, from 11.1(1.2) to 6.3(1.1) mm Hg.ml-1, (p < 0.05), and EES-single beat also decreased, from 9.6(1.0) to 7.3(1.2) mm Hg.ml-1, (p < 0.05). CONCLUSIONS: EES calculated from one beat is similar to EES determined from variably loaded left ventricular loops and responds appropriately to inotropic stimulation. This technique provides a reasonable method to calculate EES from left ventricular pressure and stroke volume without altering preload or afterload.

Analysis of cardiac assistance by latissimus dorsi cardiomyoplasty with a time varying elastance model.

OBJECTIVE: The clinical use of skeletal muscle cardiomyoplasty is limited because of its inadequate haemodynamic benefits. To facilitate experimental and clinical efforts to improve the efficacy of this technique, a mathematical model was proposed and its validity was tested in acute experiments. METHODS: The model was based on the assumption that the skeletal muscle wrapped around the heart behaves as a time varying elastance that is connected in series with another time varying elastance representing the native heart. From this model two predictions were made: (1) Skeletal muscle augments the contractility of the heart by increasing the slope (Ees) of the end systolic pressure-volume relation; (2) time varying elastance of the skeletal muscle chamber (Es(t)) can be estimated from that of the assisted heart. These predictions were examined in experiments. In nine anaesthetised, open chest dogs, preconditioned latissimus dorsi muscle was transposed to wrap the heart. Left ventricular pressure (catheter tipped micromanometer), and volume (conductance catheter) were measured while reducing the preload by vena caval occlusion to evaluate Ees with 1:2 (stimulation:heart beat ratio) stimulation of the skeletal muscle. RESULTS: With the stimulation of latissimus muscle, the end systolic pressure-volume relation was linear and Ees increased from 8.6(SEM 2.4) to 11.9(SEM 3.4) mm Hg.ml-1. Estimated Es(t) reflected the stimulation pattern and could account for the mechanism of the cardiac assistance. CONCLUSIONS: Skeletal muscle cardiomyoplasty improved the haemodynamic variable (Ees) as predicted by a mathematical model.

Ectopic production of parathyroid hormone by small cell lung cancer in a patient with hypercalcemia.

Severe hypercalcemia (serum calcium, 4.37-4.84 nmol/L) was found in a 70-yr-old man who had a small cell carcinoma of the lung with multiple metastases. The plasma immunoreactive PTH concentration was markedly elevated, as measured in three different PTH assays [N-terminal PTH, 4,650 ng/L (normal, 230-630); midregion PTH, 13,850 ng/L (normal, 180-560); C-terminal PTH, 9,900 ng/L (normal, less than 1,300)], but at autopsy the parathyroid glands were histologically normal. The PTH concentration of a liver metastasis was 503.5 ng/g wet wt (normal liver, less than 4.2-5.9), and the PTH in the tumor extract eluted at nearly the same position as synthetic human PTH-(1-84) on gel filtration chromatography. Northern blot analysis revealed PTH mRNA in the tumor as a single band of 0.9 kilobase. These results indicate that the ectopic PTH production by the lung cancer was the cause of hypercalcemia in this patient.

T-cell receptor variable gene analysis of renal allograft-infiltrating cells in biopsy specimens using a nonradioisotopic micromethod.

BACKGROUND: A sensitive micromethod for T-cell receptor (TCR) analysis is needed for clonality analysis of renal allograft-infiltrating T cells (RAITs) obtained by needle biopsy. METHODS: TCR cDNA was amplified by the anchored polymerase chain reaction and was hybridized with 28 different TCR beta variable (TCRBV) genes fixed on nylon membranes, and the percentage of each TCRBV gene was measured spectrophotometrically. RESULTS: The specificity and linearity of the hybridization technique and the constancy of the TCRBV percentages over a wide range of sample amounts were demonstrated by control experiments. Analysis of RAITs of biopsy specimens from four patients showed broad or skewed TCRBV usage, indicating the presence of polyclonal and oligoclonal RAIT populations, respectively. In one patient who received OKT3 immunosuppressive treatment, the TCRBV skewness was dramatically reduced after the treatment. CONCLUSION: We have established a powerful method for analyzing RAIT clonality, which is especially useful for monitoring RAIT dynamics after immunosuppression therapy.

Localization of a G-protein-coupled inwardly rectifying K+ channel, CIR, in the rat brain.

The cellular localization of a G-protein-coupled K+ channel, CIR, in the rat brain has been demonstrated using a CIR-specific antibody, in combination with in situ hybridization. The CIR protein and messenger RNA were found in the cerebellar cortex, hippocampal formation, olfactory system, cerebral cortex, basal ganglia, several nuclei of the lower brain stem and the choroid plexus. In contrast to the messenger RNA, which was concentrated in the cell soma, the CIR protein was found in a subset of nerve fibers and, in other cases, in axon terminals. In the cerebellar cortex and hippocampus, the CIR protein was concentrated in the axon terminals of basket cells which are known to be GABAergic interneurons. This discrepancy between the distribution of protein and messenger RNA was observed in the substantia nigra, the interpeduncular, trigeminal, hypoglossal, oculomotor and red nuclei of the lower brain stem, and the tufted and mitral cells of the olfactory bulb. These observations suggested the translocation of the CIR protein into the nerve fibers following synthesis in the cell soma. Furthermore, its specific neuronal localization, especially in GABAergic interneurons, suggested the importance of CIR in synaptic transmission in neuronal systems.

Noninvasive method to obtain input function for measuring tissue glucose utilization of thoracic and abdominal organs.

We have developed a method for the noninvasive estimation of regional tissue glucose utilization in humans that employs positron emission tomography (PET) and 2-(18F)fluoro-2-deoxy-d-glucose (FDG). Unlike other methods, the input function used in this method is obtained from the corrected time-activity curve of the descending aorta, not the left ventricle, because the descending aorta is relatively free of spillover from other organs and extends from the upper thorax to the lower abdomen. With this method the time-activity curve of the descending aorta must be corrected for the partial volume effect and the difference in counts between plasma and whole blood. Using the noninvasively obtained input function, regional tissue glucose utilization was calculated by Patlak graphic analysis. k1k3/(k2 + k3) was in good agreement with k1k3/(k2 + k3) calculated from the plasma input function by arterial sampling (r = 0.9995). These results suggest that the input function and regional tissue glucose utilization (not only of myocardium but also of other thoracic and abdominal organs) can be determined noninvasively.