Measurements of Strong-Interaction Effects in Kaonic-Helium Isotopes at Sub-eV Precision with X-Ray Microcalorimeters.

We have measured the 3d→2p transition x rays of kaonic ^{3}He and ^{4}He atoms using superconducting transition-edge-sensor microcalorimeters with an energy resolution better than 6 eV (FWHM). We determined the energies to be 6224.5±0.4(stat)±0.2(syst) eV and 6463.7±0.3(stat)±0.1(syst) eV, and widths to be 2.5±1.0(stat)±0.4(syst) eV and 1.0±0.6(stat)±0.3(stat) eV, for kaonic ^{3}He and ^{4}He, respectively. These values are nearly 10 times more precise than in previous measurements. Our results exclude the large strong-interaction shifts and widths that are suggested by a coupled-channel approach and agree with calculations based on optical-potential models.

Observation of Coulomb-Assisted Nuclear Bound State of Ξ

In an emulsion-counter hybrid experiment performed at J-PARC, a Ξ^{-} absorption event was observed which decayed into twin single-Λ hypernuclei. Kinematic calculations enabled a unique identification of the reaction process as Ξ^{-}+^{14}N→_{Λ}^{10}Be+_{Λ}^{5}He. For the binding energy of the Ξ^{-} hyperon in the Ξ^{-}-^{14}N system a value of 1.27±0.21 MeV was deduced. The energy level of Ξ^{-} is likely a nuclear 1p state which indicates a weak ΞN-ΛΛ coupling.

The chimeric protein, PEBP2 beta/CBF beta-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptional activation.

The chromosomal inversion 16(p13;q22) associated with human acute myeloid leukemia generates the chimeric PEBP2 beta/CBF beta-SMMHC gene. The PEBP2 beta/CBF beta portion of the chimeric polypeptide harbors most of the amino acid sequence of the PEBP2 beta/CBF beta protein, the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, whereas the SMMHC portion of the chimera consists of the rod domain of the smooth muscle myosin heavy chain molecule. In this study we examined the subcellular localization of the chimeric protein and its effect both on stress fibers and transcriptional activation by transfecting cDNA into tissue culture cells. The localization of the chimera was investigated by immunocytochemical staining of cells and was found to be both cytoplasmic and nuclear. One aspect of the effect of expression of the chimera was a drastic alteration of cell morphology. The cells appeared elongated and possessed long cytoplasmic processes. Double fluorescent labeling revealed disorganization of the stress fibers and an altered F-actin staining pattern in the transfected cells. Studies using a deletion mutant showed that both the PEBP2 beta/CBF beta and SMMHC domains are necessary for the induction of the morphological alteration. A significant proportion of the chimeric protein was retained in the cytoskeleton after detergent extraction of the cells and could be recuperated as a membrane fraction, suggesting that this is one of the probable sites of action of the PEBP2 beta/CBF beta-SMMHC protein. Another effect of the chimeric protein was inhibition of transcriptional activation dependent on the PEBP2/CBF binding DNA sequence. However, deregulation of PEBP2/CBF site dependent transcription by itself was not sufficient to induce cell morphological changes. Taken together, these results indicate that the PEBP2 beta/CBF beta-SMMHC chimeric protein acts at two levels, at the level of stress fiber organization and at the level of transcriptional activation. We suggest that the action of PEBP2 beta/CBF beta-SMMHC depends to a great extent on whether it is located in the cytoplasm or in the nucleus.

Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization.

A ubiquitin (Ub)/ATP-dependent proteolytic complex (26S proteasome) purified from rabbit skeletal muscle was dissociated into two subcomplexes, a 20S proteasome and a regulatory subunit complex, by preparative non-denaturing polyacrylamide gel electrophoresis (PAGE). The isolated regulatory subunit complex preparation gave a single broad band on analytical non-denaturing PAGE, and several bands ranging between 33 and 110 kDa on SDS-PAGE. This complex was found to consist of about 20 subunits on the basis of two-dimensional PAGE, the pattern of which appeared identical or very similar to that of the 33-110 kDa 26S proteasome subunits. The apparent molecular mass of the complex was estimated to be 1100 kDa by Ferguson plot analysis and also by Superose 6 gel filtration. Unlike the 26S proteasome, neither ATPase activity nor protease activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Phe-Ser-Arg-MCA, Z-Leu-Leu-Glu-beta NA, [14C]-casein, [125I]-lysozyme and Ub-[125I]-lysozyme were significantly detectable in the regulatory subunit complex. This complex was found to be capable of associating with itself in MgATP-dependent manner. These results suggest that a regulatory subunit complex dissociated from the 26S proteasome comprises all the higher molecular mass subunits of the 26S proteasome, and has no detectable ATPase and protease activities, although the homo-oligomerization occurs in an ATP-dependent fashion.

Difference between PA700-like proteasome activator complex and the regulatory complex dissociated from the 26S proteasome implies the involvement of modulating factors in the 26S proteasome assembly.

The PA700-like proteasome activator complex was highly purified from porcine erythrocytes, and its properties were compared with those of the regulatory complex disassembled from the purified 26S proteasome. The molecular mass of the PA700-like complex, which comprises 25-110-kDa subunits, was estimated to be 800 kDa by Superose 6 gel filtration. This complex showed neither ATPase activity nor peptidase activity toward Suc-Leu-Leu-Val-Tyr-MCA. Nevertheless, it was possible to make a high molecular mass complex from the purified PA700-like complex by incubating with the 20S proteasome in the presence of ATP. In contrast, the regulatory complex dissociated from the 26S proteasome did not reconstitute a larger complex under the same conditions. The subunit composition of the PA700-like complex was similar but not identical to that of the regulator complex dissociated from the 26S proteasome: the former complex had a 25-kDa subunit which is absent in the latter, whereas the latter had two or three 43-kDa subunits lacking in the former. These results indicate that the purified PA700-like proteasome activator complex is structurally and functionally distinct from the regulatory complex dissociated from the 26S proteasome, implying the involvement of modulating factors in the 26S proteasome assembly.

Different ratios in 20 S proteasomes and regulatory subunit complexes in two isoforms of the 26 S proteasome purified from rabbit skeletal muscle.

A ubiquitin/ATP-dependent proteinase complex (26 S proteasome) was highly purified from rabbit skeletal muscle. The purified 26 S proteasome easily dissociated into a 20 S proteasome and a regulatory subunit complex on non-denaturing PAGE. By using cleavable and non-cleavable cross-linkers, it was revealed that the 26 S proteasome exists in two isoforms: one (D complex) consists of the 20 S proteasome and the regulatory subunit complex in the ratio of one to two, while the other (C complex) exists in an equal molar ratio. Molecular masses of the former and the latter isoforms were estimated to be 1,700 kDa and 1,400 kDa, respectively, by gel filtration, and 2,400 kDa and 1,400 kDa, respectively, by Ferguson plot analysis. Furthermore, both isoforms efficiently hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and ubiquitin-conjugated [125I]lysozyme. These results suggest that the D and C complexes are active proteinase complexes, most probably corresponding to the dumbbell-like and mushroom-like (or space capsule-like) molecules, respectively.

Detection of cell adhesion molecules in lens epithelial cells of human cataracts.

PURPOSE: To detect the expression of cell adhesion molecules (CAMs), including beta integrins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1, leukocyte-endothelial cell adhesion molecule-2, E-cadherin, and CD44, in cultured lens epithelial cells (LECs) of human cataracts. To show that LECs attach to cells or extracellular matrix components by the detected adhesion molecules. METHODS: A circular section of the anterior capsule with attached LECs obtained by anterior capsulotomy during cataract surgery was cultured in a well of an eight-chamber slide. The LEC immediately after surgery and the cell outgrowth beyond the capsular margin at 2 weeks of culture were observed after the culture was stained immunohistochemically. Functional assays of LEC growth on collagen- or laminin-coated plates were performed in the presence and absence of the antibody blocking the detected adhesion molecules. RESULTS: beta 1 integrin, ICAM-1, and CD44 were detected in both the original specimens and the cultured cells. When the antihuman anti-beta 1 integrin monoclonal antibody (mAb), anti-ICAM-1 mAb, or anti-CD44 mAb was added at 10 micrograms/ml to the incubation medium, LEC migration and proliferation were inhibited significantly on the collagen- or laminin-coated plates. When the mAb blocking these three CAMs were added each at 1 microgram/ml, LEC proliferation also were inhibited. CONCLUSIONS: beta 1 integrin, ICAM-1 and CD44 are all involved in LEC attachment and growth on collagen and laminin in vitro. It can be assumed that these CAMs are involved in adhesion of LECs to extracellular matrix components of the lens capsule. Understanding the characteristics of the adhesion molecules in LEC may lead to the development of a new approach to inhibit secondary cataract formation.

Thoracoscopic en bloc total esophagectomy with radical mediastinal lymphadenectomy.

OBJECTIVE: Total esophagectomy with en bloc mediastinal lymphadenectomy for cancer carries a substantial morbidity and mortality rate. To investigate the feasibility of thoracoscopic technique, we carried out an extensive laboratory study. Encouraged by our excellent results, we conducted a clinical trial. METHODS: From September 1994 to September 1995, 39 patients thoracic esophageal cancer lesions not invading surrounding organs underwent total esophagectomy with mediastinal lymphadenectomy by means of thoracoscopy. Ages ranged from 47 to 86 years. The procedures were conventional except for the thoracic portion, which was performed as a thoracoscopic procedure with six trocar holes instead of thoracotomy. All harvested lymph nodes were counted for each station. Spirometric data and plethysmographically determined vital capacity were measured before and after operation for all patients. RESULTS: All procedures were accomplished as scheduled, and none was converted to open thoracotomy. The operating time was 200 +/- 41 minutes (mean +/- standard deviation). Estimated blood loss was 270 +/- 157 ml. The harvested lymph nodes numbered 19.7 +/- 11.1 per patient. Seventeen patients (45%) had positive lymph nodes. There were no in-hospital deaths within 30 days. Twenty-two patients did not require postoperative ventilatory support. Vital capacity decreased to 85% +/- 11% of the preoperative values, and forced expiratory volume in 1 second decreased to 82% +/- 16%. CONCLUSIONS: Thoracoscopic mediastinal lymphadenectomy is technically feasible, and its completeness is comparable to that of the open technique. The decline in pulmonary function is significantly less than that seen in our previous experience with the open technique.

Responses of neurons in the nucleus basalis of Meynert to various afferent stimuli in rats.

The effects of innocuous and noxious mechanical stimulation of skin, and of baroreceptor and chemoreceptor stimulation, on the activity of single neurons in the nucleus basalis of Meynert (NBM), whose axons project to the cortex, were examined in urethane-anesthetized adult rats. Most of the neurons were not significantly influenced by innocuous mechanical cutaneous stimulation or baroreceptor stimulation, while they were excited by noxious mechanical cutaneous stimulation and chemoreceptor stimulation. The NBM neurons were excited more intensely and frequently by nociceptive mechanical stimulation to a fore- or hindpaw than by that to the back or face. The function of these NBM neurons is discussed.

Nerve growth factor-mediated sexual differentiation of the rat hypothalamus.

Injection of antibody to nerve growth factor into the cerebral lateral ventricle blocked testosterone-induced behavioral defeminization of neonatal female rats. When tested as adults following ovariectomy and combined estrogen-progesterone treatment, the injected animals showed a significantly higher lordosis quotient than the testosterone-treated, normal rabbit serum-infused controls. Failure of vaginal opening and clitoral enlargement manifested the well-documented masculinizing effect of testosterone on the genitalia in the experimental as well as the control animals. Estrogen sensitivity of hypothalamic neurons which are responsible for the induction of lordosis was retained in the experimental animals. Recordings of the antidromic action potentials from neurons in the ventromedial nucleus of the hypothalamus following stimulation of the midbrain central gray revealed that estrogen decreased the antidromic activation threshold and shortened the absolute refractory period of the hypothalamic efferents along with the estrogen-induced behavioral activation in the experimental animals. In the control group, the estrogen-induced neuronal activation was lost altogether with the behavioral activation.

Morphine augments excitatory synaptic transmission in the dentate gyrus through GABAergic disinhibition.

The present study investigated the effect of morphine on synaptic transmission and long-term potentiation (LTP) in the dentate gyrus using rat hippocampal slice preparations. Field excitatory postsynaptic potential (fEPSP) and population spike (PS), evoked by stimulation of the perforant path, were recorded from the dentate molecular layer and the stratum granulosum, respectively. Following application of 10 microM morphine, PS amplitude increased gradually in 10 min and was eventually potentiated by approximately 50%. The phenomenon showed a concentration-dependent manner and was completely canceled by naloxone, a mu opioid receptor antagonist. Furthermore, morphine-induced PS augmentation was not detected in disinhibited hippocampal slices, which suggests that the inhibitory input to the dentate granule cells was required for the facilitatory effect of morphine. Neither fEPSP nor tetanus-induced LTP of PS was altered by morphine application. The data support the hypothesis that mu opioid receptor activity modulates inhibitory recurrent circuits in the dentate gyrus and thereby, indirectly plays a regulatory role for hippocampal excitatory neurotransmission.

Efficacy of long-term treatment with nipradilol, a nitroester-containing beta-blocker, in patients with mild-to-moderate essential hypertension.

The effects of long-term treatment with nipradilol, a nitroester-containing beta-blocker, on casual and 24-hour blood pressures were studied in 70 patients with mild-to-moderate essential hypertension. Antihypertensive effects of nipradilol on casual blood pressure were observed in 68% of patients. Nipradilol reduced pulse rates, but no bradycardia was observed. The usefulness of nipradilol in the present study was 65%. The results of ambulatory blood pressure monitoring indicated that nipradilol reduced systolic blood pressure more than diastolic blood pressure, and reduced blood pressure during waking more than during sleep. These results suggest that nipradilol is a safe and useful long-term antihypertensive drug in both young and older patients with mild-to-moderate essential hypertension. When administered twice daily, nipradilol is effective throughout a 24-hour period.

Electrical properties of paraventricular neurosecretory neurons with and without recurrent inhibition.

Twelve out of 32 neurosecretory neurons in the paraventricular nucleus of rats showed a silent phase following subthreshold stimulation to the posterior pituitary gland. After suprathreshold stimulation, the duration of the silent phase was significantly longer than that of the remaining 20 neurons, which did not show the silent phase at subthreshold stimulation. The latency and threshold in the former neurons were significantly longer and higher than those of the latter neurons. These data indicate a relationship between the recurrent inhibitory system and other electrical properties in the paraventricular neurons.

Estrogen excites oxytocinergic, but not vasopressinergic cells in the paraventricular nucleus of female rat hypothalamus.

Extracellular antidromic potentials were recorded from the paraventricular nucleus of the ovariectomized female rat hypothalamus following electrical stimulation of the neurohypophysis. Effects of estrogen-treatment were investigated after classifying the antidromically identified cells into tonically-firing, phasically-firing, or silent groups according to their patterns of spontaneous discharge. Estrogen significantly decreased the antidromic activation threshold and shortened the refractory period as well as the antidromic spike latency in the tonic-firing cells. We suggest that estrogen selectively and directly excited the tonically-firing, presumably oxytocinergic cells.

Hypothalamic osmoregulation for vasopressin release in streptozotocin-diabetic rats in vivo and in vitro.

The central osmoregulation mechanism for vasopressin (VP) release was studied in the streptozotocin diabetic (STZ-DM) rat. Electrical activities of the VP-producing cell in the supraoptic nucleus (SON) were recorded extracellularly and compared with those in the control rat both in vivo and in vitro. Neuronal activities in the periventricular area (PVA) were also recorded in the in vitro experiment. Hyperosmolar stimulation which was done with an intraperitoneal injection of 1.0 M NaCl (4 ml/kg) resulted in an increase in plasma osmolality both of STZ-DM (increased by 14 +/- 2 mOsml/kg H2O) and control rats (15 +/- 3 mOsmol/kg H2O). Increased plasma osmolality caused significant increase in the mean discharge rate of VP-producing cells in the control animals, but only an insignificant change in STZ-DM rats. In the hypothalamic slice preparations incubated in the artificial cerebrospinal fluid (301 +/- 2 mOsml/kg H2O), VP-producing cells in control rats increased their discharge rate linearly as the osmolality (310 +/- 2 and 320 +/- 1 mOsmol/kg H2O) or concentration (10(-8) and 10(-6) M) of angiotensin II (AGII) of the perfusate was increased stepwise, but there was no change in response to either stimulus in STZ-DM rats. On the other hand, there was no difference in sensitivity to osmolality and AGII of PVA neurons in both animal groups. These data indicate that lower sensibility to osmotic change of VP-producing cells in STZ-DM rats may depend, at least partially, upon the disturbance of osmo- and AGII-sensitivity in VP-producing cells themselves, and that these changes seem to be restricted to SON VP-producing cells.

Leumorphin, a novel opioid peptide, promotes lordosis in female rats.

The actions of leumorphin, a recently characterized endogenous opioid peptide, oppose of most opioid peptides in facilitating lordosis reflex, a major component of female sexual behavior in the rat. Maximal lordosis appeared promptly after infusion of 1 nmol leumorphin into the ventromedial hypothalamus of ovariectomized estrogen-primed rats. This facilitation lasted for as long as 5 h, unless interrupted by midbrain infusion of an antiserum to prolactin. The result is a discovery of a novel substance of remarkable strength in facilitating lordosis, an effect presumably mediated by midbrain release of prolactin.

Electrophysiological evidence for multiple sites of actions of angiotensin II for stimulating paraventricular neurosecretory cells in the rat.

Microelectrophoretically (MEPh) applied angiotensin II (AII) excited about half of the PV neurosecretory cells recorded. The excitation was blocked by MEPh applied Sar1-Ala8-AII. Intraventricular (IVT) injection of AII excited both the sensitive cells and insensitive cells to the MEPh applied AII. MEPh-applied Sar1-Ala8-AII, however, blocked the IVT AII induced excitation only in the former type of the cells.

Responses of paraventricular and supraoptic units to angiotensin II, sar1-ile8-angiotensin II and hypertonic NaCl administered into the cerebral ventricle.

Extracellular recordings of action potentials were made from neurones antidromically identified as neurosecretory cells in the paraventricular and supraoptic nuclei of urethane-anesthetized female rats. Eighty-six neurones were examined for their responsiveness to 10 ng of angiotensin II (AII) injected into the third cerebral ventricle and 78 (91%) of them increased their firing rate following the AII injection. None of the neurosecretory cells tested showed a response to the intraventricular (IVT) injection of isotonic NaCl. Thalamic neurones and non-neurosecretory hypothalamic neurones did not respond to the AII given IVT. Firing activity of 13 neurosecretory neurones was recorded during reflex milk ejection induced by suckling pups in the lactating rats. Seven of them were classified as oxytocinergic cells because they showed a burst of activity before reflex milk ejections and the remaining 6 neurones which gave no burst of firing before milk ejections were classified as nonoxytocinergic neurones. The IVT application of AII resulted in activation of all the oxytocinergic neurones and 5 of the 6 non-oxytocinergic neurones. The effect of AII on the firing of the neurosecretory cell was inhibited by the simultaneous application of Sar1-Ile8-AII (1 microgram), a competitive AII antagonist. The IVT injection of the antagonist alone inhibited the spontaneous firing of the neurosecretory cells, but it did not affect the firing of thalamic or non-neurosecretory hypothalamic neurones. Hypertonic NaCl (0.85 M NaCl, 1 mu1 IVT) also activated 13 of 20 neurosecretory cells tested. Combined application of AII and hypertonic NaCl elicited a marked potentiation of the response of neurosecretory cells to each of the stimuli. These findings indicate that AII activates neurosecretory cells stimulating specific AII receptors in the brain and that AII has a synergistic action with hypertonic NaCl. Inhibition of spontaneous activity of neurosecretory cells by a competitive AII antagonist suggests that endogenous AII may participate in the maintenance of basal activity of neurosecretory cells.

Purification and properties of the 26S proteasome from the rat brain: evidence for its degradation of myelin basic protein in a ubiquitin-dependent manner.

A ubiquitin(Ub)/ATP-dependent proteolytic complex (26S proteasome) was highly purified from the rat brain. The brain 26S proteasome consisted of 22-110 kDa subunits characteristic of the typical 26S proteasome on the basis of SDS-PAGE. The two-dimensional PAGE (NEPHGE and SDS-PAGE) pattern revealed that the pI values and molecular masses of the brain 26S proteasome subunits were similar to those of the subunits of 26S proteasomes purified from the rat liver and skeletal muscles. The enzymatic properties of the brain 26S proteasome were similar to those of the liver complex and also to the Ub-conjugate degrading activity in the cerebral cortex extract. Furthermore, it was found that the brain 26S proteasome was capable of degrading the myelin basic protein in a Ub-dependent manner. These results indicate that the brain contains the Ub-conjugate-degrading 26S proteasome, the subunit composition of which appears similar to those of the other tissues, and that the myelin basic protein may be a candidate physiological substrate for the brain 26S proteasome.