Lectin histochemistry in rat thyroid tumours

The thyroid tumours and background goiterous and adenomatous lesions induced in rats with diisopropanolnitrosamine (DIPN) plus methylthiouracil (MTU), and regenerative thyroid tissues after wounding were studied by lectin histochemistry. Ten weeks after cessation of the carcinogen treatments, carcinomas invading the surrounding tissues and blood vessels (13/20) and papillary micronodules (11/20) were formed in the thyroid tissues. In general, the carcinoma lesion was solitary, and the papillary micronodules were multiple in a single thyroid gland. Among the lectins tested, Maclura pomifera (MPA) and Solanum tuberosum (STA) showed specific binding with both carcinoma and papillary micronodule lesions, but not with the background goiterous and adenomatous lesions and regenerative thyroid tissues. The former both lesions showed higher labelling indices with BUdR or 3H-thymidine and poorer thyroglobulin accumulation than the latters, thereby indicating their enhanced proliferative capability and depressed potency of cyto-differentiation. The common cytological and histochemical properties of carcinoma lesions and papillary micronodules allow us to regard the latter as pre-invading carcinoma lesions. The lectins MPA and STA may be, therefore, used as the specific markers of malignancy in rat thyroid carcinogenesis.     

Immunochemical evidence that myosin I heavy chain-like protein is identical to the 110-kilodalton brush-border protein

In a previous study, we identified a new mammalian myosin heavy chain, termed myosin I heavy chain-like protein (MIHC), by molecular cloning of a bovine intestinal cDNA clone. In this investigation, we examined the relationship between MIHC and the 110-kDa intestinal brush-border protein, which possesses a myosin-like ATPase activity. We raised antibodies against a chemically synthesized oligopeptide representing a part of the MIHC sequence. These antibodies reacted specifically in immunoblots with the 110-kDa protein in both purified 110-kDa protein-calmodulin complex and crude microvillar protein extracts. Staining of tissue sections with these antibodies was specifically localized to the brush-border microvilli of small intestines, indicating an identical cellular localization for both MIHC and the 110-kDa protein. Furthermore, analysis of the MIHC sequence revealed two putative calmodulin-binding sites, which is consistent with the fact that the 110-kDa protein forms a complex with calmodulin. These results strongly support the conclusion that MIHC is identical to the 110-kDa protein and suggest that not only the conventional myosin system but also the MIHC (110-kDa protein)-calmodulin complex may play an important role in ATP-dependent and Ca2+-induced brush-border contraction.     

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.     

Effect of testosterone on the susceptibility of C57BL/6 mice to infection with Brugia pahangi with reference to inflammatory cell response

Effects of testosterone on the susceptibility and inflammatory cell responses of C57BL/6 mice infected intraperitoneally with Brugia pahangi larvae were examined. On day 15 postinfection, female mice showed significantly greater resistance than did males, and peritoneal cell responses (lymphocytes, macrophages, and eosinophils) were great in females. Castration of highly susceptible male mice increased their resistance and peritoneal cell responses to the level of female mice; whereas, castration of female mice did not affect the susceptibility and cell responses. Furthermore, testosterone treatment at a physiological dose in the castrated male mice or a pharmacological dose in female mice suppressed resistance and inflammatory cell responses. These results suggest that male sex hormone, testosterone, but not female sex hormone has a regulatory role in the susceptibility and cellular response of C57BL/6 mice to infection with B. pahangi, and it causes differences between sexes in susceptibility.     

Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing.

We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.     

Isolation and structure of an intrastrand cross-link adduct of mitomycin C and DNA.

A new covalent mitomycin C-DNA adduct (4) was isolated from DNA exposed to reductively activated mitomycin C (MC) in vitro. The MC-treated DNA was hydrolyzed enzymatically under certain conditions, and the new adduct was isolated from the hydrolysate by HPLC. Its structure was determined by ultraviolet and circular dichroism spectroscopy and chemical and enzymatic transformations conducted on microscale. In the structure, a single 2" beta, 7"-diaminomitosene residue is linked bifunctionally to two guanines in the dinucleoside phosphate d(GpG). The guanines are linked at their N2 atoms to the C1" and C10" positions of the mitosene, respectively. A key to the structure was a finding that removal of the mitosene from the adduct by hot piperidine yielded d(GpG); another was that the adduct was slowly converted to the known interstrand cross-link adduct 3 by snake venom diesterase and alkaline phosphatase. Adduct 4 represents an intrastrand cross-link in DNA formed by MC. Of the two possible strand-polarity isomers of 4, 4a in which the mitosene 1"-position is linked to the 3'-guanine of d(GpG) is designated as the proper structure, on the basis of the mechanism of the cross-linking reaction. The same adduct 4 was isolated from poly(dG).poly(dC), synthetic oligonucleotides containing the GpG sequence, and Micrococcus luteus and calf thymus DNAs. The relative yields of interstrand and intrastrand cross-links (3 and 4) were determined under first-order kinetic conditions; an average 3.6-fold preference for the formation of 3 over that of 4 was observed. An explanation for this preference is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early nuclear events of in vitro fertilization in the domestic fowl (Gallus domesticus)

In vitro fertilization in the domestic fowl (Gallus domesticus) was investigated by observation of the early nuclear events. Ova retrieved from the fimbria following ovulation were inseminated in vitro with 10(6)-10(7) spermatozoa in Dulbecco's modified Eagle's medium (DMEM) for 10 min and then further incubated in DMEM + albumen for 1, 2, 3, or 4 hr. These eggs were histologically examined by epifluorescent microscopy after staining with 4',6'-diamidino-2-phenylindole (DAPI). Nuclei of spermatozoa at various stages of transformation were observed in the ova incubated for 1-3 hr. Close pairing of two pronuclei, presumed to be male and female juxtaposition, was detected in ova incubated for 4 hr. These data provide direct evidence for the in vitro fertilization of fowl eggs and suggested that the early process of in vitro fertilization is comparable to that of in vivo fertilization.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Genetic Mapping of Four Mouse bHLH Genes Related toDrosophilaProneural Geneatonal

It has been shown that mammalian neurogenesis is partly controlled by multiple basic helix–loop–helix (bHLH) genes, as inDrosophila.Recently, mouse homologs ofDrosophila atonal,a proneural gene encoding a bHLH protein required for chordotonal organ and photoreceptor development, have been characterized to obtain further insights into the molecular nature of mammalian neurogenesis. Here, to assess their potential involvement in genetic neural disorders, we have determined genetic map positions for four mouseatonal-related genes,Atoh1, Atoh2, Atoh3,andNdrf,which encode MATH-1, MATH-2, MATH-3, and NDRF, respectively. Interspecific backcross analysis indicated thatAtoh1andAtoh2were located in separate positions of Chr 6 and thatAtoh3andNdrfwere mapped to Chr 10 and Chr 11, respectively. Thus, these structurally related genes are located separately on multiple chromosomes.