Spin-dependent acoustic response in the nonunitary A1 and A2 phases of superfluid 3He under high magnetic fields.

The transverse acoustic impedance of superfluid ^{3}He was measured in the A1 and A2 phases up to 13 T to investigate the surface states in nonunitary superfluids. The temperature dependence of the impedance was much larger in the A1 phase than in the A2 phase. This nonsymmetric behavior indicates that momentum exchange with walls for spin-down surface states is quite different from that for spin-up surface states. The spin-dependent response might be a reflection of an essential feature of the nonunitary states where gap amplitudes depend on spin states. Weak-coupling theories ignore any spin-dependent processes and do not account for the nonsymmetric behavior.

Magnetic response of odd-frequency s-wave Cooper pairs in a superfluid proximity system.

We investigate the magnetic response of a dirty-normal-Fermi-liquid-spin-triplet-superfluid proximity system consisting of liquid 3He and aerogel. In contrast to bulk superfluids, Pauli spin susceptibility in the proximity system exceeds its normal-state value locally around the interface. This enhanced Pauli paramagnetism originates from odd-frequency s-wave pairing arising due to spatial inhomogeneity. A characteristic observable signature of the paramagnetic effect can be found in the spin susceptibility temperature dependence.

New anomaly in the transverse acoustic impedance of superfluid 3He-B with a wall coated by several layers of 4He.

We measured the transverse acoustic impedance of superfluid 3He-B with a wall coated by several layers of 4He. The coating is known to enhance the specularity in quasiparticle scattering by the wall. We found a new anomaly, a bump and a peak, in the temperature dependence of the transverse acoustic impedance. This agrees with a theoretical calculation using a partially specular wall boundary condition. The new anomaly is shown to arise from a change in the surface density of states by coating and the scattering of thermally occupied surface bound states to other states. The change is towards the density of states of Majorana cone in the specular limit.

A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice

We describe two recessive alleles of a rice heterochronic gene, plastochron1-1 (pla1-1) and pla1-2, that reduce the length of the plastochron to approximately half that of the wild type. Because the onset of the reproductive phase in pla1 was not temporally affected, the number of leaves produced in the vegetative phase was nearly twice that produced in the wild type. Panicle development was severely disturbed in pla1 mutants. In pla1-1, many primordia of primary rachis branches were converted into vegetative shoots. These ectopic shoots repeated the initiation of panicle development and the conversion of primary rachis branches into shoots. In the weak allele pla1-2, however, only the basal one or two primordia developed as vegetative shoots, and the remaining primordia developed to produce a truncated panicle. These results indicate that both vegetative and reproductive programs are expressed simultaneously during the reproductive phase of pla1; however, the degree varied depending on the strength of the allele. Accordingly, pla1 is a heterochronic mutation that extends the vegetative period. The shoot apical meristem of pla1 was larger than that of the wild type, although the shape was not modified. An in situ hybridization experiment using the histone H4 gene as a probe revealed that cell divisions are accelerated in the pla1 meristem. The PLA1 gene is considered to regulate the duration of the vegetative phase by controlling the rate of leaf production in the meristem.

Immunohistochemical detection of aberrant p53 expression in hepatocellular carcinoma: correlation with cell proliferative activity indices, including mitotic index and MIB-1 immunostaining.

We analyzed the p53 expression immunohistochemically in 50 specimens of hepatocellular carcinoma (HCC) using two monoclonal antibodies (DO7 and PAb1801) and one polyclonal antibody (CM1), which recognize both wild and mutant type p53 proteins and can be used for paraffin-embedded sections. Fifteen of the 50 HCC specimens (30%) showed p53 expression localized at tumor nuclei, and this expression was significantly more frequent in HCCs with histologically lower differentiation. Except for serum titers of alpha-fetoprotein, the p53 expression had no statistically significant correlation with clinicopathological parameters, including hepatitis virus infection, tumor size, and background liver diseases. Conversely, the cell proliferative activities of tumor cells as assessed by mitotic index and immunostaining for MIB-1 were well correlated with the grade of histological differentiation. Moreover, MIB-1 immunostaining was shown to be useful in distinguishing well differentiated HCC from hepatocytes in chronic liver diseases. It also was shown that p53 expression was strongly associated with cell proliferative activity. Our results indicate that p53 expression takes place in the late stage of tumor progression and is related to the high malignant potential of HCCs.

A genetic and physical map of the region containing PLASTOCHRON1, a heterochronic gene, in rice ( Oryza sativa L.).

The rice heterochronic gene plastochron1, pla1, shows shorter plastochron and ectopic expression of the vegetative program during the rice reproductive phase resulting in aberrant panicle formation. A genetic and physical map was constructed to isolate the causal gene for the pla1 syndrome. Small-scale mapping was carried out to determine the approximate map position of the pla1 locus, and then a high-resolution genetic map was made for pla1-1, one of the pla1 alleles, using an F(2) population comprising 578 pla1-1 homozygous plants. In a high-resolution genetic map, the pla1-1 locus was found to map between RFLP markers C961 and R1738A on chromosome 10, within a 3.6-cM genetic distance. A physical map encompassing the pla1-1 locus was constructed by overlapping Bacterial Artificial Chromosome (BAC) clones through chromosome walking. PCR-based RFLP markers from BAC-end clones were developed and mapped relative to the pla1 locus. Physical map construction using BAC clones indicated that a BAC clone, B44A10 (167-kb), contained the pla1 locus within 74-kb corresponding to a 0.52-cM genetic distance. Gene prediction of 74-kb region carrying the pla1 locus suggested several candidate genes for the pla1 gene. Identification of a candidate gene for pla1 will be made by sequence analysis of allele variation and cDNA screening.

Morphological abnormalities in the hippocampus of the weaver mutant mouse.

The lamination of the hippocampus in the homozygous B6CBA weaver mouse (wv/wv) was compared with that in normal B6CBA littermates (+/+) and C57BL/6J mice using Nissl and Timm's staining. In Nissl-stained preparations, the normal littermates exhibit a compact, regular arrangement of pyramidal cells in area CA3 of the hippocampus. In contrast, in homozygous weaver mutant mice, the pyramidal cell layer of area CA3 frequently appears to be thicker than normal with an apparent increase of neuropil, as evidenced by the presence of cell-free spaces within the layer. Also, small ectopic clusters of pyramidal cells and sometimes the subdivision of the pyramidal cell layer into 2 or 3 layers were found throughout the dorsoventral extent of the hippocampus. In Timm's stained preparations of the normal mouse hippocampus, two clearly separated bundles of axons were seen emerging from the hilus: one bundle running above the pyramidal cell layer of area CA3 (i.e., the suprapyramidal mossy fiber layer, SPMFL), and the second bundle running below the pyramidal cell layer (i.e., the infrapyramidal mossy fiber layer, IPMFL). In contrast, in some homozygous weaver mice, the origin of the mossy fiber bundles is clearly different from normal; specifically, mossy fibers emerge in a diffuse fashion from the area between suprapyramidal and infrapyramidal mossy fiber layers. In other weaver mice, short, discontinuous bundles diverge from the infrapyramidal mossy fiber layer and invade the thickened pyramidal cell layer. In addition, ectopic pyramidal cells are situated below the IPMFL in area CA3. The morphological changes observed in hippocampus of weaver mutants are likely to be secondary to a more basic genetic defect.

The abnormal distribution of mossy fiber bundles and morphological abnormalities in hippocampal formation of dreher(J) (dr(J)/dr(J))mouse.

The organization of pyramidal cells and mossy fibers in the hippocampal formation of homozygous dreher(J) mutant mice was investigated using Timm's and Golgi methods. Five clear abnormalities were found: (1) some pyramidal cells were located below the infrapyramidal mossy fiber layer, (2) mossy fibers emerged in diffuse fashion from between the suprapyramidal and infrapyramidal mossy fiber layers, and their fibers invaded within the pyramidal cell layer, where they traveled as 3-6 small, usually quite short, bundles, (3) some normally situated pyramidal cells had unusual contacts with mossy fibers at two or three places on their apical and/or basal dendrites, (4) some normally situated pyramidal cells had abnormal dendritic trees typified by the occurrence of fine-caliber dendritic branches extending out of the apical dendrite or the apical portion of the soma, and (5) a few Timm positive fibers extending from the dentate hilus to the dentate molecular layer in both dreher(J) and control mice were observed. These abnormalities indicate that in the hippocampal formation a variety of cell populations and neuronal circuits can be indirectly modified by the dreher mutation.

Staining of semithin tissue sections embedded in HPMA, quetol 523 and MMA.

Various tissues fixed in a mixture of formaldehyde and glutaraldehyde, and embedded in an improved 2-hydroxypropyl methacrylate mixture were employed for studying the fine structures of cells and tissues by light microscopy. The embedding mixture contained Quetol 523 and methyl methacrylate as a plasticizer without a cross-linker. The catalyst was QCU-1. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogeneous block from which it was easy to cut sections of 1-2 microns in thickness. A wide variety of stains have been employed with such sections and those reported here are hematoxylin-eosin, Azan and PAS. There was excellent preservation of alkaline phosphatase activity. A method of poststaining immunoperoxidase labeling was also applied to the mouse pancreas and examples of staining with insulin are included.

Correlative light and electron microscopy of the same sections embedded in HPMA, Quetol 523 and MMA.

Semithin sections, cut from tissues stained with acid and basic dyes after embedding in 2-hydroxypropyl methacrylate, Quetol 523 and methyl methacrylate, showed cytoplasmic components at a high resolution by light microscopy. These same sections could then be viewed, after osmium tetroxide, uranyl and lead staining, by the electron microscope. These sections had a number of inherent advantages: they could be observed with a light microscope; they facilitated analysis of cellular structures in the identical sites, and they were frequently the optimum thickness to provide three-dimensional information. We clearly established the structural detail of this same-section correlative light-electron microscopy approach by showing that the coloured materials observed in such sections of cells followed the distribution of fine structures within the same sections as determined by electron microscopy. In some instances the fidelity of the correlation between the distribution of the coloured area and cytoplasmic components in identical cells of the same section revealed significant details which could not visualized in thin sections. This technique, therefore, provided a simple and useful solution to many problems that require the localization of cellular components in identical cells selected previously by light microscopy.

Studies on thick sections of the nucleus of mouse Sertoli cells using an electron microscope operating at 300 kV.

Examination of the three-dimensional structure of the Sertoli cell nucleus from mouse testes was performed under a high voltage electron microscope operating at 300 kV. Using an en bloc staining method along with fixation by osmium tetroxide and embedding in a mixture of Quetol 651, NSA and MNA, the structures of the nucleus were stained at a high contrast and satisfactory preservation was achieved, thus allowing their study at a high resolution within thick sections. Nuclear components could be observed clearly in 2-3 microns-thick sections of embedded material. Typical three-dimensional configurations of nucleoli and associated bodies were indicated. Thick sections permitted the observation that two or three pernucleolar bodies are usually attached on each side of the nucleoli or form a triangular shape of different sizes of vacuolar structures within the bodies. Stereoscopic observations also revealed overlapping of perinucleolar bodies and nucleoli and suggested the complexity of the components of perinucleolar and intranucleolar chromatin.

Histochemical demonstration of heavy metals in the hippocampal formation embedded in Quetol 523M.

To facilitate improvement of investigations on the distribution of mossy fibers in the hippocampal formation, a method is described using Timm's stained preparations after methacrylate embedding with the hydrophilic resin, Quetol 523M. Fixation with a mixture of formaldehyde and glutaraldehyde yielded satisfactory staining results and good structural preservation. During the course of histochemical experiments employing Timm's staining, examinations revealed that sulfide silver reaction products were consistently present in both the mossy fibers themselves and their terminals associated with the dendrites of pyramidal cells in tissue sections of 1-2 microns in thickness. The results obtained also revealed that variations of the mossy fiber system occurred in the neurological mutant mouse dreher (dr). The bundles of mossy fibers forming the intrapyramidal synaptic field may be considered to reflect genotype-dependent differences in the mutation. The present method is adequate for allowing the histochemical demonstration of mossy fibers and their giant boutons by light microscopy.

Use of semithin sections embedded in a water-miscible methacrylate for light microscopy of central nerve tissues.

Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogenous block from which it was easy to make sections 1.5 microns thick. Staining could be localized far more precisely in these sections than in paraffin sections owing to the thickness of the semithin sections and to the excellent structural preservation of cellular components.

Light and electron microscopic observations of glycogen in semi-thin sections employing GMA, Quetol 523 and methylmethacrylate.

The use of improved GMA-Quetol 523 or GMA-Quetol 523 methyl methacrylate (MMA) mixture as an infiltration medium for the histochemical demonstration of glycogen has been devised to facilitate embedding, sectioning and staining. An improved method of infiltration uses such mixtures with a double weight of QCU-1 as a catalyst. Since the double weight of catalyst prevents interference by bleeding of picric acid from tissue blocks into the resin mixtures, improved mixtures penetrate readily and completely into the tissue fixed in picric acid-formaldehyde-glutaraldehyde solution (PAFG). PAFG solution has been found suitable for the histochemical fixation of glycogen in semithin sections. In hepatocytes specific reaction products appeared a deep reddish-purple after periodic oxidation and Schiff's reaction (PAS reaction), which was lost in previous digestion by means of salivary deastase. Glycogen can be identified as a PAS positive area in semi-thin sections 0.2-0.3 micrometers thick under the light microscope. Identical reactions sites of such sections show a high contrast in electron microscopy without any staining by either heavy metals or osmium tetraoxide vapor. It was therefore demonstrated that total glycogen produced after PAS reaction was revealed distinctly with deep contrast precipitates. This method provides significant morphological data for the histochemical localization of glycogen.

Observation on backscattered electron image (BEI) of a scanning electron microscope (SEM) in semi-thin sections prepared for light microscopy.

In order to examine semi-thin section for light microscopy with the backscattered electron mode (BE mode), identical sites in tissue sections were comparatively observed with both light microscopy and BE mode. Tissue blocks (ca. 3 X 3 X 1 mm) were fixed in glutaraldehyde or combined formaldehyde-glutaraldehyde solution. After dehydration in alcohol, they were embedded in Kushida's GMA-Quetol 523. 1.0 micron sections on glass slides coated with indium oxide were stained with hematoxylin-eosin or toluidine blue or by the Giemsa method, and then treated with osmium tetroxide vapor or aqueous KMnO4 solution or uranyl acetate-lead citrate solution. The identical places of such sections could be examined with the accelerating potential of 6 kV and the probe current of 8 X 10(-10) A using a JSM-35C SEM with BEIS BE detector. Photographs were taken with the 2500-line resolution cathode ray tube and the time of exposure was 100 sec. The sections were placed at a distance of 5mm from the BE detector. BE images from osmium tetroxide vapor staining showed a distinctly improved contrast especially when the sections were previously stained with hematoxylin and eosin. The cellular structure was clearly demonstrated under the electron microscope in the BE mode. Identical sites in tissue samples could be compared exactly with both light and electron micrographs.

Localization of periodate-Schiff reactive glycosaminoglycans in semi-thin sections embedded in GMA-Quetol 523-MMA--application of a method for correlative light and electron microscopy of identical sites.

A correlative light and electron microscope method in which semi-thin sections of embedded tissue were treated with periodate acid and Schiff's reagent (PAS) was used to determine the precise localization of PAS reactive substance. Small blocks of tissue specimens were fixed with aldehyde mixtures. After dehydration, the blocks were embedded in a modified mixture of glycol methacrylate (GMA), Quetol 523, methyl methacrylate (MMA) and QCU-1. Semi-thin sections, 0.2-0.3 micron thick, were stained by the PAS reaction, followed by counterstaining with hematoxylin if necessary. It was found that PAS reaction products representing the specific sites for glycosaminoglycans and glycoproteins were seen in both light and electron microscopy. In the control experiments the specificity of the reaction was confirmed. The granules of goblet cells were well stained and contrasted by the reaction materials. The basement membrane and the microvilli of the epithelial cells appeared as the staining layer. In the spermatocytes the reaction products were demonstrated in the Golgi apparatus, acrosomal vesicles and head cap. The results indicated that the PAS deposits became electron dense when the embedding matrix had a low electron scattering property. Using this method of preparing semi-thin sections, a comparative study of the localization of glycosaminoglycans was performed.

PAS staining of eosinophils in semi-thin sections of bone marrow embedded in glycol methacrylate.

The usual periodic acid-Schiff (PAS) reaction of glycosaminoglycans is applicable to paraffin embedded material. A modification for water-miscible methacrylate embedded tissue suitable for correlative light and electron microscopic studies, which makes it possible to find the same stained cell in a semi-thin tissue section, is described. Eosinophil leukocytes in semi-thin sections from bone marrow were verified by electron microscopy after staining with the PAS reaction. Immature eosinophil leukocyte granules reacted with and without previous salivary treatment. This method facilitates the search for localization of glycosaminoglycans in scattered blood cells.

[A case of advanced gastric remnant carcinoma with Virchow's metastasis treated with neoadjuvant chemotherapy (low dose CDDP + 5-FU) followed by surgical resection].

The patient was a 64-year-old woman. At hospitalization she had gastric remnant carcinoma with Virchow's and paraaortic lymph node metastases, extensive local infiltration and obstructive jaundice. The lesions were considered nonresectable, and the patient was placed on neoadjuvant chemotherapy consisting of low-dose CDDP and 5-FU, which resulted in the disappearance of Virchow's and paraaortic lymph node metastases. She was considered to have a partial response (PR) and underwent lower esophageal resection, total remnant gastrectomy and splenectomy. Eight months after surgery, however, she died of disseminated carcinomatosis of bone marrow. Since this therapy was associated with only slightly adverse events (< or = Grade 1), this treatment modality appears to be safe. However, further studies will be necessary to identify what type of recurrence is responsive to this therapy and to evaluate its effect on patient survival.

[Percutaneous ethanol injection (PEI) for small hepatocellular carcinoma].

Percutaneous ethanol injection (PEI) was applied to 162 lesions in 133 patients with hepatocellular carcinomas (HCC) 3 cm or less in diameter (small HCC) between Aug. 1983 and Apr. 1992. Histological findings in resected specimen for which PEI had previously been performed before surgery showed that PEI could completely necrotize an HCC of 32 mm in diameter. The antitumoral effect by PEI could correctly be evaluated by enhanced CT. The 1-, 3-, 5- and 7-year survival rates after PEI calculated by the Kaplan-Meier method were 95.9%, 60.5%, 36.9% and 21.7%, respectively. The survival rate of post-PEI patients with I or II grade in clinical stage was better than that of those with III. Recurrence occurred in hepatic areas different from the original lesion in 27.8% in one year and 63.6% in three years after PEI, rates quite similar to those of recurrence after surgical resection for HCCs of 3 cm or less in diameter. For such recurrence, PEI alone was then repeated in half of the 76 patients. Complications caused by PEI were not serious and did not necessitate intensive care. Because of its anti-tumor therapeutic effect and minimal damage to the liver, PEI might be considered a viable alternative to surgery for most patients with small HCC.

[An angiographic study on the pathological features of multiple hepatocellular carcinoma].

Seventy-one patients with untreated hepatocellular carcinoma (111 tumors) were studied angiographically to investigate the pathological features of multiple carcinoma. The 111 tumors comprised 23 lesions resected from 14 patients and 88 lesions measuring 3 cm or less in diameter detected in 57 patients who did not undergo resection. Hemodynamically, major lesions exhibited an increase in frequency of tumor angiogenesis and intensity of tumor stain with an increase in diameter. Analysis of angiographic features of tumors measuring 2 cm or less in diameter revealed a greater vascularity in intrahepatic metastatic foci than in primary foci, demonstrating a difference between them. When multiple tumors were classified into the isolated, concentric or disseminated type in terms of the pattern of their distribution, their angiographic findings suggested that min or lesions of the concentric or disseminated type might represent local metastases spread from the primary focus, and that those of the isolated type might represent multicentric occurrence in the liver.