Search

We are developing position-sensitive transition-edge sensor (TES) microcalorimeters (PoSTs) to detect gamma rays up to a few MeV. Each gamma-ray PoST consists of a long absorber with a TES on each end and works as a 1D imaging spectrometer. We fabricated PoSTs with 0.5 × 0.5 × 18.8 mm lead absorbers. We irradiated two PoST devices with gamma rays from a Cs-137 source. Gamma-ray pulses of the PoSTs show a strong correlation between the pulse height and the rise time. We divided the pulses in the 662 keV line into 12 groups after sorting them by rise time to determine effective pixels. We modeled the PoST device and compared the average pulses of the 12 effective pixels to numerical simulations. The actual pulses and simulated pulses were in good agreement. [ABSTRACT FROM AUTHOR]

Prompt gamma activation analysis using a focused thermal neutron guided beam at JAEA JRR-3M was applied to the determination of B in ceramic certified reference materials (BAM CRM S-003 Silicon Carbide Powder and NMIJ CRM 8004-a Silicon Nitride Powder). Cl and Si were used as internal standards to obtain linear calibration curves of B. The analytical result of B in BAM CRM S-003 was in good agreement with the certified value. The relative expanded measurement uncertainties ( k = 2) were 4.8% for BAM CRM S-003 and 4.9% for NMIJ CRM 8004-a. [ABSTRACT FROM AUTHOR]

Radiation-induced cavernous malformations are rarely reported, and most cases have been children. We describe two adult patients with cavernous malformation after irradiation for astrocytoma. Magnetic resonance (MR) imaging, at their ages of 53 years, showed a cavernous malformation in the irradiated field 26 and 10 years after resection and irradiation, respectively. Cavernous malformations were confirmed by the histopathological examination in the both cases. Radiation-induced cavernous malformations are rare in adult patients with astrocytoma. One reason why we found two such cases was that these patients had been successfully treated for astrocytoma and had long follow-up periods. [ABSTRACT FROM AUTHOR]

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells. [ABSTRACT FROM AUTHOR]

Summary ¶Background. We evaluated cerebral metabolic change during brain hypothermia with intravascular perfusion of cooled crystalloid solution using an extracorporeal cooling-filtration system and cerebroprotective effects of this hypothermia on brain injury in an animal model. Method. Microdialysis probes were implanted into the bilateral parietal cortices. A cold-induced brain injury was produced behind the microdialysis probe on the right parietal cortex. Immediately after injury in the cooled group (n=9), Ringer’s solution cooled to 5 °C was infused into the right vertebral artery after occlusion of the bilateral common carotid and the left vertebral arteries. Excessive fluid was ultrafiltrated by a dialyzer. Brain temperature was maintained at about 20 °C for 60 minutes. In 7 dogs, three neck arteries were occluded for 60 minutes after injury without cooled fluid infusion. The extracellular concentrations of glutamate, lactate, and pyruvate were measured serially for 180 minutes after injury. Findings. Extracellular glutamate concentrations in the cooled group did not increase, while there was a significant increase in the injured hemisphere as compared to the uninjured hemisphere in the non-cooled group (P<0.05). Extracellular lactate concentrations increased slightly after occlusion in both groups. The depth of cortical injury was limited in the cooled group, but extended into the white matter in the non-cooled group up to 240 minutes after injury. Interpretation. Occlusion of three main arteries induced ischaemia under critical threshold in canine brains. Under this condition, intravascular cooling with crystalloid solution suppressed accumulation of extracellular glutamate and reduced tissue damage in the early phase after cold-induced brain injury, as cerebroprotective effects. This information suggests that a method employing brain hypothermia via intra-arterial cooling with an extracorporeal cooling-filtration system has potential to achieve successful, safe, selective brain cooling. [ABSTRACT FROM AUTHOR]

Background: We investigated whether diffusion-weighted echo-planar magnetic resonance (MR) imaging can help differentiate intraductal mucin-producing tumors of the pancreas from other cystic lesions.Methods: Diffusion-weighted echo-planar MR imaging was performed in patients with mucin-producing tumors (n = 19), pseudocysts (n = 9), chronic pancreatitis with diffuse main pancreatic dilatation (n = 5), and serous cystadenomas (n = 2). Images were obtained with diffusion sensitizing gradients of 30, 300, and 900 s/mm2. The apparent diffusion coefficient (ADC) was calculated.Results: The mean (+/- standard deviation) ADCs of mucin-producing tumors (2.8 x 10(-3) mm2/s +/- 1.0 x 10(-3)), pseudocysts (2.9 x 10(-3) mm2/s +/- 1.2 x 10(-3)), dilated main pancreatic duct in chronic pancreatitis (3.3 x 10(-3) mm2/s +/- 1.2 x 10(-3)), serous cystadenomas (2.9 x 10(-3) and 2.6 x 10(-3) mm2/s), and cerebrospinal fluid (3.5 x 10A(-3) mm2/s +/- 1.1 x 10(-3)) were not statistically different.Conclusion: It is difficult to differentiate between mucin-producing tumors and other cystic lesions by ADC measurements when using diffusion-weighted echo-planar MR imaging. [ABSTRACT FROM AUTHOR]

We investigated six Japanese autopsy cases of Pick's disease with Pick bodies (PDPB) both clinically and pathologically, and examined the distribution of their cerebral cortical lesions using hemisphere and/or bisphere specimens. The lesions were classified into three categories (slight, moderate, and severe). Two patients with a clinical diagnosis of primary progressive apraxia and of slowly progressive aphasia had speech apraxia as their initial signs, and the other two patients were suspected as having Alzheimer's disease, with the clinical diagnosis of the remainder two patients being presenile dementia and depression, respectively. Extrapyramidal signs, believed to be rare in PDPB, were present in four patients. Severe lesions were multicentrically present in the cerebral cortices of all six cases. In two patients with speech apraxia, severe lesions were seen in the primary motor area, which generally has not been regarded as an "atrophic center" in Pick's disease. Furthermore, in a patient with depression, severe lesions were more widespread in the convexity than in the orbital region of the frontal lobe. The parietal lobes, including the postcentral gyrus usually believed to be spared in Pick's disease, were severely involved in three patients. We postulate that the clinical features of PDPB have a much wider spectrum than previously believed. In addition, we believe that the distribution of the cerebral cortical lesions in PDPB is more widespread than previously assumed, and that clinical manifestations of PDPB depend to some extent on the topographic distribution of the cerebral cortical lesions. [ABSTRACT FROM AUTHOR]

We report the radiologic findings of sclerosed hemangioma (SH), a rare variant of hepatic hemangioma. Dynamic contrast-enhanced computed tomography showed a hypodense mass in the liver with delayed enhancement. T2-weighted magnetic resonance imaging showed the mass as hypointense in relation to cerebrospinal fluid. The final diagnosis of SH was made pathologically. Although SH is rare, understanding its radiologic appearance is important to avoid unnecessary surgery and should be included in the differential diagnoses of hepatic lesion with delayed enhancement. [ABSTRACT FROM AUTHOR]

We present a case of adrenal metastasis from clear-cell renal cell carcinoma in which presence of a small amount of fat was shown on chemical shift gradient-echo magnetic resonance imaging. Radiologists should be aware that signal loss of the adrenal tumor on out-of-phase gradient-echo images does not always suggest the diagnosis of benign adenoma, particularly in patients with a history of renal cell carcinoma. [ABSTRACT FROM AUTHOR]

We present two cases of abdominal cystic tumors containing small amounts of fat in their septa. Although the final pathologic diagnoses of these tumors were cystic lymphangioma and angiomatosis, the computed tomographic and magnetic resonance imaging features were almost identical and indistinguishable; a purely cystic mass around the region of the pancreas head associated with little mass effect on the surrounding organs and septa containing a radiologically evident fatty component. Radiologists should be aware of these two entities as differential diagnoses of abdominal cystic masses containing small amounts of septal fat. [ABSTRACT FROM AUTHOR]

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarum polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14–19 are transcribed. [ABSTRACT FROM AUTHOR]

Two ftsZ homologues were isolated from the unicellular primitive red alga Cyanidioschyzon merolae ( CmftsZ1 and CmftsZ2). Phylogenetic analysis revealed that CmftsZ1 is most closely related to the ftsZ genes of α-Proteobacteria, suggesting that it is a mitochondrial-type ftsZ gene, whereas CmftsZ2 is most closely related to the ftsZ genes of cyanobacteria, suggesting that it is a plastid-type ftsZ gene. Southern analysis indicates that CmftsZ1 and CmftsZ2 are both single-copy genes located on chromosome XIV in the C. merolae genome. Northern analysis revealed that both CmftsZ1 and CmftsZ2 are transcribed, and accumulate specifically before cell and organelle division. The results of Western analysis suggest that CmFtsZ1 is localized in mitochondria. [ABSTRACT FROM AUTHOR]

The generative cell at anthesis in the mature pollen grain of Erythrina crista-galli (Fabaceae) was examined by 4,6-diamidino-2-phenylindole(DAPI)-fluorescence microscopy using the squash method. An unequal, polarized distribution of DNA-containing organelles (plastids and/or mitochondria) within the generative cell was observed in every mature pollen grain examined. Polarization of DNA-containing organelles is obvious when generative cells are freed and assume a spherical shape soon after microspore mitosis, as revealed by fluorescence-microscopic observations of specimens embedded in Technovit 7100 resin and thin-sectioned at different developmental stages. Early establishment of polarized localization of organelles in young generative cells of E. crista-galli and maintenance of this unequal distribution until pollen maturation strongly suggests that the organelles may still be clustered at pollen mitosis. Production of a dimorphic pair of sperm cells, as has been reported in Plumbago zeylanica, was observed in some pollen tubes germinated in vitro. The differentiation of the two sperm cells is discussed in relation to possible preferential double fertilization in angiosperms. [ABSTRACT FROM AUTHOR]

We report two cases in which the MRI appearances mimicked the dural tail sign; a glioma extending into the subarachnoid space, and a meningioma extending to the subdural space. They indicate that tumour invasion into the subarachnoid or subdural space, should be considered when prominent linear enhancement is observed along the dura mater adjacent to tumours. [ABSTRACT FROM AUTHOR]

Probiotics are used for various intestinal diseases. However, their effects on gut epithelial cell proliferation have not been investigated. We administered 10(7) colony-forming units of Lactobacillus casei or Clostridium butyricum, or no probiotics (control) by gastric intubation once a day for seven days to rats fed an elemental diet. We estimated the crypt cell production rate of the jejunum, ileum, cecum, and distal colon. We also quantified cecal bacteria. Both probiotics increased the crypt cell production rate of the jejunum and ileum by 25-40%, of the cecum by 70%, and of the distal colon by more than 200% compared with control. Only minor variance in the cecal bacterial composition existed among the three groups. Probiotics enhanced gut epithelial cell proliferation in rats fed an elemental diet. [ABSTRACT FROM AUTHOR]

When BY-2 cultured tobacco ( Nicotiana tabacum L.) cells were transferred to auxin-depleted culture medium containing cytokinin (benzyladenine, 1 mg/l), the starch content per cell started increasing from 18 h of culture and amyloplasts had formed by 48 h. Pulse-treatment of the cells with actinomycin D and cycloheximide for the first 12 h (or longer) of culture significantly decreased the cellular starch content after 48 h, whereas the starch content did not decrease significantly when the cells were released from the inhibition within 6 h. This suggests that nuclear gene expression necessary for amyloplast formation begins 6–12 h after the transfer. Immunoblotting analysis of the accumulation of ADP-glucose starch glycosyl transferase (starch synthase) supported this inference. [ABSTRACT FROM AUTHOR]

The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum. [ABSTRACT FROM AUTHOR]

A phase II study of a new combination therapy was performed using intraarterial (i.a.) cisplatin and Adriamycin in combination with i.v. methotrexate and vincristine for 27 patients with invasive urinary bladder carcinoma of stages T2-3NOMO, and the therapeutic effects were assessed. Methotrexate (20 mg/m2) was given i.v. on days 1,15, and 22, and vincristine (0.7 mg/m2) was injected i.v. on day 2 before i.a. infusion therapy and on days 15 and 22. The i.a. chemotherapy was performed after both superior gluteal arteries had been embolized using 3- or 5-mm stainless-steel coils. A mixture of cisplatin (50-70 mg/m2) and Adriamycin (20 mg/m2) was infused i.a. via both internal iliac arteries over a period of 20-30 min. Angiotensin II (mean dose, 21 micrograms) was simultaneously infused i.a. in 15 of 27 patients. In 24 of the 27 patients, at least 2 cycles of full-dose chemotherapy were completed. The dose was decreased in the remaining 3 patients because of their poor health status and advanced age. Among the 27 patients, 9 and 14 had complete (CR) and partial responses (PR), respectively; 3 manifested no change (NC), and 1 had progressive disease (PD). The objective response rate (CR+PR) was 85.2%. Among the 27 patients staged T2-3 NOMO, 6 (CR, 1; PR, 5) underwent total cystectomies and 18 (CR, 8; PR, 8; NC, 2) had transurethral resection of a bladder tumor (TUR-Bt) or partial resections following chemotherapy. The remaining 3 diminished-dose patients had no surgery. Of the 27 patients, 22 were alive after a median follow-up period of 21+ (range, 7-48+) months. No significant side effect was observed except for lower extremity paresthesias in 5 patients (18.5%). These results point to the effectiveness of this therapy and to the possibility of urinary bladder preservation in patients with invasive, advanced urinary bladder cancers. [ABSTRACT FROM AUTHOR]

Mature Jasminum officinale and J. nudiflorum pollen grains were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. The pollen grains were found to be trinucleate, and the sperm cells in both species contained a large number of epifluorescent spots that corresponded to cytoplasmic DNA aggregates (nucleoids). The nucleoids of J. nudiflorum were observed to be dimorphic under the epifluorescence microscope, indicating that the sperm cells might contain both plastid and mitochondrial DNA. The nucleoids of J. officinale presented a similar appearance when stained with DAPI, but electron microscopic examination of the sperm cells revealed that they contained both plastids and mitochondria. When analyzed by DNA immunogold electron microscopy, gold particles were detected on both plastids and mitochondria. These findings demonstrated the preservation of plastid and mitochondrial DNA in mature sperm cells and thus the potential for biparental cytoplasmic inheritance in J. officinale and J. nudiflorum. [ABSTRACT FROM AUTHOR]

We investigated the pathophysiological and morphological responses of anaesthetized rats to fluid percussion brain injury generated by an original midline fluid percussion injury device. Following different grades of trauma, lCBF was measured continuously in the right parietal cortex through a burr hole using laser Doppler flowmeter, and physiological parameters were monitored. Pathological changes also were evaluated microscopically. During the first 2 hours following trauma, we found four patterns of cerebral circulatory responses. Little measurable pathophysiological response occurred after percussion pulses of less than 1.33 atmospheres (atm). In animals subjected to pulses of greater than 4.30 atm, lCBF increased synchronously with blood pressure, and then both parameters decreased continuously until death. In animals subjected to pulses of 1.53 to 2.33 atm, trauma produced a transient increase in 1CBF with no synchronous rise in blood pressure. In animals subjected to pulses of 2.70 to 3.87 atm, lCBF increased synchronously with blood pressure immediately following the injury, but had decreased markedly by 60 seconds and remained below the pre-injury baseline. Blood pressure recovered to baseline within 4 minutes of the injury. The transient increase in lCBF occurred within 5 seconds following percussion pulses of greater than 1.53 atm and appeared to be independent of the rise in systemic blood pressure. Apnoea occurred in animals subjected to pulses of greater than 1.53 atm, and the duration of apnoea and mortality rate correlated with the magnitude of the applied injury. A power decrease in the electroencephalogram post-injury and a delay in its recovery, both depended on the magnitude of the injury with few regional differences in the beta-2 band power. The distribution and extent of blood-brain barrier disruption and small haemorrhages also correlated with the magnitude of the injury. The number of neurons decreased significantly in both hippocampi by 2 weeks following moderate trauma. The four patterns of lCBF changes demonstrated in the present study, as well as the other responses to injury, may be useful for studying graded models of various diffuse brain injuries. [ABSTRACT FROM AUTHOR]

Twenty-three patients with aneurysmal subarachnoid haemorrhage (SAH), who showed an ST segment elevation in their electrocardiograms (ECG), were examined. There were 12 males and 11 females, with a mean age of 61 years. The clinical condition on admission was Hunt and Kosnik grade II in four, III in seven, IV in one, and V in 11 patients. Computerized tomography (CT) also revealed many cases of diffuse, thick SAH or intracerebral or intraventricular haematoma. Laboratory examinations including serum electrolyte, pH, and PaO revealed no abnormalities that might have influenced the ECG. Elevation in the levels of myocardial enzymes in serum was observed in two of the nine patients examined, although the elevation was only slight in one of them. Echocardiography, which was performed on several occasions on all patients, and cardiac catheterization, which was performed on eight patients, revealed a reduction in the motion of the left ventricular apex that was synchronous with ST segment elevation. This is the first report about these phenomena. No abnormalities were observed in the coronary artery. The elevated ST segment was normalized within one week in all patients, accompanied by normalization of the apical wall motion recorded on echocardiograms. In four patients, however, T wave inversion accompanied the improvement of the ST segment and was normalized within three months after the onset. These results suggest that ST segment elevation in the acute stage of SAH reflects transient cardiac dysfunction rather than myocardial injury. In some patients, however, the elevated serum levels of myocardial enzymes or T wave inversion suggested the presence of myocardial injury. Close follow-up seems to be necessary in such cases [ABSTRACT FROM AUTHOR]

"Two-route chemotherapy" (TRC) using cis-diamminedichloroplatinum(II) (DDP) and its antidote, sodium thiosulfate (STS), combined with the angiotensin II (AT-II)-induced hypertension method was evaluated for its efficacy against peritoneally disseminated tumors in rats. A bolus i.p. injection of DDP (15 mg/kg) was given 1 min after the initiation of an AT-II (16.5 micrograms/kg) i.v. infusion lasting 11 min. Immediately after the termination of the AT-II infusion, 1,580 mg/kg STS was injected i.v. over a further 5 min. This modified TRC significantly improved the antitumor effect, evaluated by survival (increase in life span, 273%), compared with that achieved with other treatments, as follows: 15 mg/kg DDP i.p. and the concomitant i.v. infusion of 1,580 mg/kg STS (conventional TRC), 153% increase in life span; 5 mg/kg DDP i.p. with or without AT-II i.v. (167% and 107% increases in life span, respectively). As an index of nephrotoxicity, blood urea nitrogen (BUN) levels seen after modified TRC (21.1 mg/dl) were as low as those observed after conventional TRC (19.1 mg/dl), despite the postadministration of STS, and were much lower than those seen after DDP alone or DDP plus AT-II (35.6 and 35.7 mg/dl, respectively). Further evaluation of the effectiveness of modified TRC using various doses of DDP gave similar results. The feasibility of the administration of STS 10 min after DDP treatment was explained by the significant inhibition of DDP delivery to the kidney during the AT-II-induced hypertension. Thus, TRC combined with AT-II has a superior therapeutic effect against peritonitis carcinomatosa induced in rats. [ABSTRACT FROM AUTHOR]

We studied the effect of "two-route chemotherapy" (TRC) with intra-arterial (IA) neocarzinostatin (NCS) and IV N-(2-mercaptopropionyl)-glycine (tiopronin), its antidote, on rat limb tumors. Chemotherapy experiments were carried out on day 9 after the inoculation of 10(6) syngeneic transitional carcinoma cells into the hind limb in female Wistar King A rats. In the group given TRC, 3500 units/kg NCS and 800 mg/kg tiopronin were given via the femoral artery and the femoral vein, respectively. The antitumor effect was evaluated by the tumor weight on day 12 after the treatment. Compared with the weight of tumors in untreated controls, TRC reduced tumor weight to one-tenth, while 700 units/kg IA NCS alone reduced tumor weight to one-third and 700 units/kg systemic NCS alone reduced tumor weight to three-fourths of the control weight. In the group given TRC, WBC and nucleated bone marrow cells were completely protected and loss of body weight was slight. [ABSTRACT FROM AUTHOR]

The mode of inactivation of cis-diamminedichloroplatinum(II) (DDP) in the bloodstream and protection from its toxicity by sodium thiosulfate (STS) were investigated in rabbits. Plasma ultrafiltrate in rabbits given 5 mg/kg DDP IV and various excess molar ratios of STS IV were assayed for the active platinum levels with a new microbiological assay system using an E. coli strain. The active platinum species in the plasma were inactivated completely by co-administration of a 400-fold excess of STS IV. The rabbits were almost completely protected against both BUN increase and body weight loss normally caused by DDP when 400-fold doses of STS were given. Diuretic effects were also observed. Our data provide evidence for the basis of optimum use of STS to protect against DDP toxicity. [ABSTRACT FROM AUTHOR]

The behavior of nucleoids during the leucoplast division cycle in the epidermis of onion ( Allium cepa) bulbs was investigated using DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) staining. The leucoplast was morphologically amoeboid and continuously changed its shape. A dumbbell-shaped leucoplast divided into two spherical daughter ones by constriction in the middle region of the body. Leucoplasts contained 4-10 mostly spherical, oval, partly rodand dumbbell-shaped nucleoids which were dispersed within the bodies. The proportion of one DNA molecule of a T4 phage particle to the small leucoplast nucleoid in the grain density of negative film was 1 to 0.91. Comparison of the present result and another groups' biochemical results suggested that a small leucoplast nucleoid contains one DNA molecule. The dumbbell-shaped leucoplast probably before division contained about twice as many nucleoids as the spherical leucoplast after division, and each half of the dumbbell contained about half the number of nucleoids. Nucleoids increased in number with growth of the leucoplast. The behavior of nucleoids during the leucoplast division cycle in onion bulbs was basically similar to that during the chloroplast division cycle in higher plants and green algae, which was previously reported ( Kuroiwa et al. 1981 b). [ABSTRACT FROM AUTHOR]

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated. [ABSTRACT FROM AUTHOR]

In the primitive red alga Cyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study. C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules of C. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against a C. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin “immature” contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments. [ABSTRACT FROM AUTHOR]

A novel procedure is described for fluorescence staining of microbodies, which can be applied quickly and easily. We developed this technique of microbody staining with the unicellular red alga Cyanidioschyzon merolae. Cyanidioschyzon merolae only contains a single chloroplast, mitochondrion, and microbody per cell, and the mitotic cycle and the organelle division cycle are easily synchronized. Knowing that the concentration of H2O2 in the microbody is higher than it is in the cytosol and other cell components, we attempted to visualize the microbody by using fluorescence microscopy to detect H2O2. Brilliant sulfoflavin (BSF), used for detecting Fe2+ in analytical chemistry, fluoresces when it reacts with Fe2+ and H2C2. We were able to specifically stain microbodies with BSF, under acidic conditions (pH 3.0 or pH 2.5) with blue-light excitation. Using this procedure, we observed division of the microbody and the effect of aphidicolin on the microbody. We also discovered that microbody division is regulated by the cell nucleus and follows division of the cell nucleus. [ABSTRACT FROM AUTHOR]

The behavior of organelle nucleoids in the generative cell was examined at the second (pollen grain) mitosis by epifluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) in Oenothera biennis. The O. biennis generative cell contained a large number of organelle nucleoids distributed randomly in the cytoplasm before mitosis. The epifluorescence images of the nucleoids could be classified distinctly into two groups which corresponded to plastid nucleoids (pt-nucleoids) and mitochondrial nucleoids (mt-nucleoids). Discrimination between pt- and mt-nucleoids was carried out with the aid of DNA immunogold electron microscopy. At metaphase, both pt- and mt-nucleoids migrated to the pole regions of the generative cell. After mitosis, organelle nucleoids in both of the sperm cells scattered in the cytoplasm again. A quantitative examination of pt-nucleoids on 202 pairs of sperm cells showed that the leading sperm cell (Svn) contained 0–39 pt-nucleoids (19.0 ± 7.4) and the trailing sperm cell (Sua) contained 0–40 pt-nucleoids (15.4 ± 6.5). For mt-nucleoids, examination of 28 pairs of sperm cells showed that Svn contained 5–32 mt-nucleoids (14.5 ± 6.8) and Sua contained 6–30 mt-nucleoids (13.4 ±7.5). These results showed that (1) the number of organelle nucleoids per sperm cell varied considerably in the cells studied; (2) quantitative difference in pt- and mt-nucleoids between Svn and Sua could occur in some gametophytes studied; but (3) it was unlikely that there was any pre-differentiational cytoplasm localization and essential sperm heteromorphy with respect to organelle nucleoid content in the gametophyte population. [ABSTRACT FROM AUTHOR]

In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen of Pelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells of P. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids in P. zonale. [ABSTRACT FROM AUTHOR]

It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red alga Cyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase α, using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin on C. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3′-dihexyloxacarbocyanine iodide (DiOC6; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division in C. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA. [ABSTRACT FROM AUTHOR]

We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria from Physarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer. [ABSTRACT FROM AUTHOR]

The preferential development of giant mitochondria and their nuclei (nucleoids) in the egg cells of Pelargonium zonale Ait. during megasporogenesis and megagametogenesis was examined by fluorescence microscopy, after Technovit embedding and 4′,6-diamidino-2-phenylindole (DAPI) staining, fluorimetry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction of mitochondrial nuclei (mt-nuclei). Reproductive cells during the megaspore mother cell, meiosis, tetrad, and functioning megaspore stages contained many small mitochondria with characteristic, uniformly DAPI-stained mt-nuclei about 0.3 μm in diameter, containing a small amount of DNA (0.3 Mbp). During formation of the 2-, 4-, and 8-nucleate embryo sac, mt-nuclei did not markedly change in shape or DNA content. When the embryo sac formed and differentiation of each cell began, mitochondria and their nuclei in the egg cell took on a small ring or string-like shape. Accompanying the maturation of the embryo sac, they underwent progressive enlargement and gradually altered to long thick strings, or stacks of concentric or half concentric rings. By flower opening, they have developed to an extremely large size. One of these stacks of mt-nuclei was reconstructed in three dimensions; each ring in the stack was cup- or plate-shaped; 5 to 10 rings made up the stack, though each remained discontinuous from the others. From serial sections, we counted 44 mitochondria in one egg cell. Fluorometry using VIMPICS revealed that DNA amount within the stacked mitochondrion increased to 40 times that of the megaspore mother cell stage; a single stack of mitochondria contained 340-1700 Mbp DNA; which means that one egg cell contains at least 15000 Mbp mt-DNA, a value greater than the cell-nuclear DNA content. [ABSTRACT FROM AUTHOR]

FdUrd (5-fluorodeoxyuridine), a specific inhibitor of thymidylate synthase, was used to study the relationship between reproductive processes in chloroplast and nucleocytoplasmic compartments of the chlorococcal alga Scenedesmus quadricauda. The courses of DNA replication and nuclear division in both the compartments were followed in populations synchronised by the alternation of light and dark periods. DAPI-staining of DNA-containing structures was used for their visualisation and quantification. In contrast with cellular reproductive events, those in chloroplasts were not substantially affected by the presence of FdUrd (25 μg/ml). It was shown that FdUrd specifically blocked nucDNA replication but not ptDNA replication. Thus, cells which had attained commitment to ptDNA replication, fission of pt-nuclei and chloroplast kinesis triggered and terminated these processes while the corresponding cellular processes were blocked. The courses of reproductive processes in chloroplasts were also substantially unaffected in cells grown in the presence of FdUrd for the whole cell cycle. This provided evidence that attainment of commitment to and termination of the entire sequence of reproductive events, including chloroplast fission, were controlled by different mechanisms than the reproductive processes in the nucleocytoplasmic compartment. [ABSTRACT FROM AUTHOR]

DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga, Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them. [ABSTRACT FROM AUTHOR]

We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3′-dihexyloxacarbocyanine iodide (DiOC) and 4′,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 ( Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated. [ABSTRACT FROM AUTHOR]

We present strong electron microscopic evidence that the mitochondrial-dividing ring (MD-ring) forms as a closed ring about 50 nm wide and 10 nm thick, at the contact point where the micro-body attaches to the mitochondrion. This ring forms in the cytoplasm around an equatorial plane perpendicular to the major axis of a mitochondrion. As the MD-ring increases in both width and thickness, the mitochondrion becomes dumbbell-shaped with a narrow interconnecting isthmus. Then, by successive contractions of the ring, the dumbbell-shaped mitochondrion separates to generate two daughter mitochondria. We also observed formation of an electron dense plastid-dividing ring (PD-ring) during plastid divisions. We noted too the behaviour of the MB in relation to the contraction of MD-rings and PD-rings. [ABSTRACT FROM AUTHOR]

We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture of Physarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells of P. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster. [ABSTRACT FROM AUTHOR]

To study the mechanism of condensation of dispersed plastid (pt) nucleoids into a single pt nucleoid with aging of the cells of Chlamydomonas reinhardtii, two mutants, designated cond-1 and cond-2, were isolated. A plastid of a wild type cell, 6.5 μm in diameter, contained ten dispersed spherical pt nucleoids within one week of culture on an agar plate. At about one week of culture, the cell number was saturated and pt nucleoids began to associate with each other, condensing into a single pt nucleoid at three weeks of culture. In contrast, cond-1 and cond-2 cells, which had about 20 and 45 pt nucleoids and whose cell diameters were 7.8 and 9.5 μm at one week of culture respectively, still had about 10 and 20 pt nucleoids at even 7 weeks of culture. Doubling times of the three cell types were similar. From genetic analysis, each of the two mutants had one gene mutation. The two mutations are probably linked. The measurement of O evolution showed that the two mutations did not affect the photosynthetic system. Lipid contents of the two mutant cells were clearly higher than that of wild type cells. The role of a higher number of pt nucleoids is probably to increase the activity of lipid and/or membrane synthesis for lipid storage. [ABSTRACT FROM AUTHOR]

The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived from Nicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 μm, and 60% of them ranged from 7 to 11 μrn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition. [ABSTRACT FROM AUTHOR]

The first identification of a mitochondria-dividing ring (MD-ring), which is located in the cytoplasm near the outer envelope membrane at the constricted isthmus of dividing mitochondria in the red alga Cyanidioschyzon merolae, is reported. The MD-ring is about 50 nm wide and 10 nm thick at early stage of mitochondrial constriction and is a somewhat electron-dense circular bundle. The MD-ring is believed to be essential for the division of mitochondrion (mitochondriokinesis) since the ring appears at the equatorial region of the mitochondria just before the initiation of mitochondrial division and can be observed throughout mitochondrial division. The MD-ring has features comparable to that of the plastid-dividing (PD) ring. [ABSTRACT FROM AUTHOR]

The amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) in Cyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system. C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×10kbp, 1.6×10kbp, and 5.0×10kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those of Cyanidium caldarium RK-1 and C. caldarium Forma A. The ρ values of cl-DNA and cp-DNA of C. merolae were about 1.716 and 1.709, respectively. The order in density was different from that of C. caldarium Forma A but very similar to that of C. caldarium RK-1. However, the restriction patterns of cp-DNA in C. merolae differed from those of C. caldarium RK-1. [ABSTRACT FROM AUTHOR]