Occurrence of differentiated keratin peptide(K1) in cultured human squamous cell carcinomas.

To date, the largest keratin peptide(K1, 68 KD) has been absent in cultured human squamous cell carcinomas. Using a low salt aqueous solution, not containing high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two dimensional polyacrylamide gel electrophoresis, immunological techniques and Northern blot analysis, we demonstrated K1 peptide in two kinds of cultured human squamous cell carcinomas. Until now keratin extraction has been done using high salt/Triton X-100 solution during which K1 peptide may be removed together developed an affinity with the buffer. Many investigators may have therefore overlooked K1.     

Photolabeled sites with a tetrodotoxin derivative in the domain III and IV of the electroplax sodium channel.

Forty three percent of the labeled sites, at least, in the electroplax sodium channel with a photoactivable tetrodotoxin derivative were identified by probing protease-digested labeled fragments with several sequence-directed antibodies. They are located in the loop between segments S5 and S6 of domain IV, as well as the region containing transmembrane segment S6 and adjacent extracellular and cytoplasmic sequences in domain III. No photolabeled fragments were detected in the corresponding region of domain I. These results suggest that C-11 of tetrodotoxin where the photoreactive moiety is attached orients to the region between S5 and S6 in domain III and IV. Probable orientation of the tetrodotoxin molecule in sodium channels is considered by taking together with the recent report of the site-directed mutagenesis.     

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

A point mutation in exon 3 (His 107→Tyr) in two unrelated Japanese patients with carbonic anhydrase II deficiency with central nervous system involvement

We have analyzed two unrelated Japanese patients with carbonic anhydrase II deficiency born to consanguineous parents. We have identified the same mutation as that reported to be homozygous in a Belgian family and compound heterozygous in an American family. It comprises to C-to-T transition that results in the amino acid substitution of Tyr (TAT) for His (CAT) at position 107. This point mutation creates an AccI site that can be conveniently screened by the polymerase chain reaction/restriction fragment length polymorphism method using a restriction enzyme for gene tracking. Our patients exhibit severe mental retardation, not seen in the Belgian and American patients. Received: 23 November 1994 / Revised: 22 May 1995     

The role of β- and α-adrenoreceptors on blood flow and temperature of brown adipose tissue and involvement of nitric oxide in their effects

1. o

2. 1. Isoproterenol increased interscapular brown adipose tissue (BAT) blood flow and temperature.

3. 2. Phenylephrine increased BAT temperature, but did not affect blood flow. The effect was completely suppressed by N^ω-nitro- -arginine methyl ester ( -NAME), an inhibitor of endogenous NO synthesis.

4. 3. Isoproterenol plus phenylephrine increased BAT blood flow and temperature earlier than isoproterenol alone.

5. 4. CL316,243 increased BAT blood flow and temperature. These effects were also inhibited by -NAME.

6. 5. These data suggest that BAT blood flow as well as thermogenesis is regulated mainly by β-adrenoreceptors and partly by α₁-adrenoreceptors, and NO may be a common mediator for their regulations.

     

Expression of Dictyostelium early gene, dutA, is independent of cAMP pulses but dependent on protein kinase A

Abstract The untranslatable, RNA polymerase II-dependent gene ( dutA ) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA , requires protein kinase A for its expression.     

Failure to Reject an Allografted Tumor after Elimination of Macrophages in Mice

After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (MΦ)-rich, non-T, non-NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the MΦs in the rejection of allografted Meth A cells and characterized the MΦs in comparison with other well-known MΦs. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the MΦ-rich population. In addition, the lymphocyte-rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the MΦ-rich or the T cell-depleted MΦ-rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of MΦs by in vivo application of dichloromethylene diphosphonate-containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of MΦ is the effector cell essential for the rejection. In contrast to other well-known MΦs, the cytotoxic activity against Meth A cells was cell-to-cell contact dependent and soluble factor (e.g., NO and TNF-α) independent. Moreover, the cytotoxic activity of the MΦs (H-2^b) against ⁵¹Cr-labeled Meth A (H-2^d) cells was inhibited by the addition of unlabeled H-2^d, but not H-2^a, H-2^k or H-2^b, lymphoblasts as well as Meth A cells, implying the specific interaction of the MΦs with H-2^d cells.     

Bilateral giant myelolipoma in the adrenal of a cotton-top tamarin (Saguinus oedipus)

Abstract: A rare case of bilateral adrenal myelolipomas in a female cotton-top tamarin is reported. Large bilateral masses in the adrenal glands were composed of mature adipose cells containing varying amounts of hematopoietic cells of the myeloid, erythroid, and megakaryocyte series. The gross and histologic features of this case closely resemble human “giant” adrenal myelolipomas.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.