Preparation and Characterization of Dextran Magnetite-Incorporated Thermosensitive Liposomes: An on-line Flow System for Quantifying Magnetic Responsiveness

Purpose. Dextran magnetite (DM)-incorporated thermosensitive liposomes, namely thermosensitive magnetoliposomes (TMs), were prepared and characterized in order to investigate their possibility for magnetic drug targeting. Methods. TMs containing calcein were prepared at various DM concentrations by reverse-phase evaporation of dipalmitoylphosphatidylcholine (DPPC). They were evaluated for their physicochemical properties including size, DM capture, magnetite distribution within liposomes, and temperature-dependent calcein release. Moreover, a novel on-line flow apparatus with a sample injector, a coil of tubing placed in an electromagnet, and a fluorescence detector was developed for quantifying the magnetic responsiveness of TMs. This device allowed us a real-time measurement of percentage holding of TMs by magnetic field. Results. Due to water-soluble property of DM, higher contents of magnetite up to 490 mg per mmol DPPC were successfully incorporated into the liposomes with DM than with conventional magnetite (Fe₃O₄). Thermosensitivity and lipid integrity of TMs were not influenced by inclusion of DM. Using the on-line flow system, percentage holding of TMs by magnetic field was shown to vary with several factors; it increases as the magnetic field strength increases, the fluid flow rate decreases, the magnetite content increases, and the liposome concentration increases. Typically, at 490 mg incorporated magnetite per mmol DPPC, 0.5 ml/min-fluid flow rate, and high magnetic field strength (10 kiloGauss), approximately 100% of TMs were found to be held. Conclusions. The TMs were suggested to be useful in future cancer treatment by magnetic targeting combined with drug release in response to hyperthermia.     

Comparison of larvicidal, adulticidal and acaricidal activity of two geometrical butylidenephthalide isomers

Insecticidal and acaricidal activities of two geometrical isomers, (E)- and (Z)-butylidenephthalide isolated from Angelica acutiloba, against larvae and adults of fruit fly (Drosophila melanogaster), cat fleas (Ctenocephalides felis) and house dust mites (Dermatophagoides farinae and Tyrophagus putrescentiae) were investigated and compared with that of positive controls. (E)- and (Z)-Butylidenephthalide exhibited 50% lethal concentration (LC50) values of 2.07 and 0.94 micromol/ml of diet concentration against larvae of D. melanogaster, respectively. This indicated that two isomers of butylidenephthalide have geometrical stereoselectivity for larvicidal effect. Even though both (E)- and (Z)-butylidenephthalide also showed potent adulticidal and acaricidal activity against adults of D. melanogaster and two mites, there was no significant difference between two isomers. Insecticidal activity of both (E)- and (Z)-butylidenephthalide toward adults of C. felis was not detected even at the maximum concentration of 200 microg/cm2.     

BRD4 Bromodomain Gene Rearrangement in Aggressive Carcinoma with Translocation t(15;19)

Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescence in situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicate BRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent the BRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa.     

AN ELECTRON MICROSCOPIC EXAMINATION OF AGE-RELATED CHANGES IN THE RAT LIVER. The Influence of Diet

The influence of age and diet on the ultrastructure of hepatocytes is reported. The following dietary manipulations were investigated: Group 1, fed ad libitum a diet containing 21% protein; Group 2, fed a similar diet but restricted to 60% of the intake of Group 1 from 6 weeks of age onwards; Group 3, restricted from 6 weeks to 6 months of age and thereafter fed ad libitum; Group 4, restriction started at 6 months of age; Group 5, fed ad libitum a diet containing 12.6% protein. In all groups the size of hepatocytes was found not to increase during adult life. The size of hepatocytes in Groups 2 and 4 was the same as or larger than that of the other groups; thus food restriction resulted in a decreased number of hepatocytes. Changes in the structure of some organelles and the accumulation of lipofuscin granules occurred with advancing age and the extent of these age-related changes was less in Groups 2 and 4 than in the other groups. These morphologic findings in conjunction with our previously reported metabolic findings provide a new view of the action of food restriction on the aging process. ACTA PATHOL JPN 38: 1119∼1130, 1988.     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.     

An autopsy case of hepatic sarcomatoid tumor: Immunohistochemical comparison with a sarcomatous component of hepatocellular carcinoma

A case of primary hepatic tumor exclusively composed of malignant cells with sarcomatous features is described and compared immunohistochemically with two cases of hepatocellular carcinoma (HCC) with a sarcomatous component. More than 30% of HCC cells were positively stained with anti-cytokeratin (CAM5.2), anti-albumin, anti-fibrinogen and anti-α₁-antitrypsin antibodies, and some with anti-epithelial membrane antigen. The present sarcomatoid tumor and the sarcomatous component with HCC showed similar immuno-histochemistry; many tumor cells were strongly immuno-reactive for vimentin and some positive for cytokeratin, albumin, fibrinogen and u,-antitrypsin. Other immunohistochemical markers, indicating specific differentiations to lineage of macrophages, muscle cells, glial cells, endothelial cells and so forth, were not detected in sarcomatous tumor cells of all cases. These findings suggest that the present sarcomatoid tumor would belong to an anaplastic sarcomatous variant of HCC.     

SECRETORY GRANULES IN BENIGN AND MALIGNANT GLUCAGONOMAS OF THE PANCREAS

Two cases of glucagonoma, one benign and the other malignant, were presented. Benign glucagonoma in a 29-year old man with multiple endocrine neoplasia type 1 was composed largely of tumor cells with secretory granules ranging from 139 to 417 nm in diameter identical to A cell granules. There were a few tumor cells which contained no A cell granules but smaller granules of approximately 166 nm diameter similar to those of pancreatic polypeptide-containing cells. Radioimmunoassay of the tumor extract showed 319 μg/g wet weight of glucagon and 0.72 μg/g wet weight of pancreatic polypeptide. Malignant glucagonoma in a 34 year-old man was a massive tumor of 7x6x5 cm replacing the tail and body of the pancreas with multiple metastases. The tumor contained 0.2 μg/g wet weight of glucagon and 0.065, ug g wet weight of vasoactive intestinal peptide. The electron microscopic examination revealed that the tumor cells had variable numbers of atypical secretory granules measuring 110 to 200 nm in diameter different from A cell granules. An analysis of plasma glucagon by the gel filtration technique showed the heterogeneity of glucagon molecules indicating the presence of large glucagon. Atypical secretory granules in malignant glucagonoma were considered to represent immature granules containing the precursor or intermediate of glucagon.     

Osteogenesis bioassay and immunohistochemical and radioisotopic studies of parotin,parotid gland extract,and subunit

Bioassay data support the hypothesis that the salivary gland participates in endocrine regulation of the development and maintenance of connective tissues. Epiphyseal cartilages become dystrophic in young growing rats after the resection of all major salivary glands. A subunit obtained from parotin, an extract of bovine parotid glands, contains the active agent for the presumed endocrine function of the salivary gland. Daily injections of 3 mg/rat of parotin or the subunit allow normal epiphyseal endochondral osteogenesis in salivary gland-deprived rats. The active agent appears to be secreted by the salivary acinar cells and resorbed through the striated ducts. Pancreatic islets and striated ducts of salivary glands share immunohistochemical activities for insulin, glucagon, and the subunit of parotin. Hyperglycemia and hypocholesterolemia occur in intact rats given 5 mg/day of parotin for 30 days. Together these data suggest endocrine functions of the salivary gland and possible interactions between the pancreatic islet and salivary gland.     

Computer Imaging Analysis of Glomerular Extracellular Components in Patients with IgA Nephropathy

Computer imaging analysis was used for quantitative evaluation of the extents, amounts and distributions of glomerular extracellular components, such as the 7S and NC 1 domains of type IV collagen, laminin (LN), fibronectin (FN) and IgA, in glomeruli from patients with IgA nephropathy. Renal biopsy specimens from 13 patients with IgA nephropathy were incubated with mouse monoclonal antibodies against the FN or non collagenous (NC 1) domain of type IV collagen or polyclonal antiserum against the LN or 7S domain of human type IV collagen, and then stained with appropriate dilutions of FITC labeled anti mouse Ig antisera. Marked staining of the 7S or NC 1 domain of type IV collagen, LN or FN was detected in the glomerular capillary walls and/or mesangial areas in patients with IgA nephropathy. In particular, a prominent increase of FN was observed in the subendothelial regions of glomerular capillary walls, i.e. mesangial interposition, in the moderate or advanced stage of IgA nephropathy. Therefore, computer imaging analysis was shown to be useful for the quantitative determination of such components distributed in glomeruli from patients with IgA nephropathy. Acta Pathol Jpn 39: 296 305, 1989.     

The 5D4 antibody (anti-cyclin D1/D2) related antigen: Cytoplasmic staining is correlated to the progression of gastric cancer

In order to clarify the relationship between cyclin D1 and 02 (CD1/CD2) overexpression and progression, 191 gastric cancer cases (81 early and 110 advanced cancers) were investigated using the 504 monoclonal antibody for both CD1/CD2 in immunohistochemistry. 5D4 immunoreactlvity was noted in 68 (35.6%) cases, staining being restricted to the nucleus In 27 (14.1%) cases, the cytoplasm in 34 (17.8%) cases, and its presence in both the nucleus and cytoplasm in seven (3.7%). Cases demonstrating cytoplasmic positiv-ity, including both positive cases, were significantly more frequent in advanced cancers (P = 0.010), those having lymph node metastasis ( P = 0.004) and cases showing cancer invasion of vessels (P = 0.00S), although no relatlon to histological malignant grading was apparent. In contrast, cases of nuclear positivity behaved no differently from 5D4-negative cases. Statistics showed a trend where survival in patients was worse in the cytoplasm-positive cases than the cytoplasm-negative group. However, multivariate analysis revealed no Independent statlstical significance in the cytoplasmic positivity of prognosis. Additional studies using DCS-6 antlbody for CD1 and 617 antibody for CD2, suggest that nuclear staining of 5D4 indicates the presence of CD1 but cytoplasmic staining is derived from an antigen that is related to CD2. In conclusion, the present results indicate that the accumulation of CD2 in the cytoplasm may play some role In the progression of gastric cancers but not prognosis; however, CD1 overexpression is not linked to either.