Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Correction of congenital microtia using the tissue expander

We attempted auricular reconstruction using Radovan-type inflatable silicone expanders in six children and one adult, with the complete hypoplastic, the conchal remnant, and constricted type of microtia. Ear frameworks, including the helix, anthelix, concha, and tragus, were prepared using autologous rib cartilage. Based on the surface area of the normal adult auricle, the silicone expander was tentatively shaped and sized into a rotated semiellipse and expanded with 70 cc saline. Auricular reconstruction on the framework was completed at the time of insertion in four of the seven patients, requiring no elevation of the ear. The reconstructed auricle was satisfactory in both color and texture and had nearly normal sensation. Mild complications were noted in three of the seven patients. However, no resorption of the inserted rib cartilage has been observed 14 months to 2 years and 5 months after the operation. Slight shrinkage of the expanded skin was noted in each patient.     

Cloning and sequence analysis of a cDNA encoding the Rieske iron-sulfur protein of rat mitochondrial cytochrome bc1 complex

We have isolated a cDNA clone for the Rieske iron-sulfur protein of rat cytochrome bc1 complex, by screening a rat liver cDNA expression library using antiserum directed against the corresponding protein of bovine. The amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that the mature polypeptide of the rat protein consists of 196 amino acid residues with a molecular weight of 21,465, and that it is formed as a precursor with an amino-terminal extension. Northern blot analysis indicated that rat liver possibly contains different sizes of mRNAs for the Rieske iron-sulfur protein, and Southern blot analysis demonstrated that rats and mice possess a single gene for this protein.     

Flavonoids inhibit the expression of heat shock proteins

Cells exposed to several forms of stress, such as heat shock, transiently synthesize a group of proteins called heat shock proteins (hsps). Although many stressors other than heat shock are known to induce hsps, inhibitors of hsp expression have never been reported. Here we show that quercetin and several other flavonoids inhibit the synthesis of hsps induced by heat shock in two human cell lines, Hela cells and COLO320 DM cells. Quercetin inhibited the induction of hsp70 at the level of mRNA accumulation. This is the first report to describe the inhibition of hsp expression by reagents.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III

The ubiquinone-binding protein (QP-C) is a nuclear-encoded component of ubiquinol-cytochrome c oxidoreductase in the mitochondrial respiratory chain and plays an important role in electron transfer as a ubiquinone-QP-C complex. We obtained a partial cDNA for rat liver QP-C by screening a lambda gt11 rat liver cDNA library using antiserum directed against bovine heart QP-C. Using this cDNA as a probe, a cDNA clone was isolated from a human fibroblast cDNA library by colony hybridization. The total length of the cloned cDNA was 518 base pairs with an open reading frame of 333 base pairs. The 111-amino acid sequence deduced from the nucleotide sequence of the cDNA is 85% homologous to that of bovine QP-C and contains only a single additional amino-terminal methionine. This implies that the human QP-C is synthesized without a presequence which is required for import of most nuclear-encoded mitochondrial proteins into mitochondria.     

Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum. apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins

Two putative serum precursors which cross-react with antiserum against murine senile amyloid protein (ASSAM) were isolated from the high density lipoprotein (HDL) of normal mouse serum. Apolipoproteins designated "apoSASSAM-1" and "apoSASSAM-2" have the same molecular weight as tissue amyloid fibril protein. ApoSASSAM-1 and apoSASSAM-2 migrate to an intermediate position between apoA-I and apoC on alkaline-urea polyacrylamide gel electrophoresis and are present mainly in HDL apoproteins and to a slight extent in very low density lipoprotein apoproteins when compared to apoC. ApoSASSAM-1 and apoSASSAM-2 are polymorphic; there are two apparent isoproteins of apoSASSAM-1 with isoelectric points of 4.72 and 4.79 and two major isoproteins of apo-SASSAM-2. Subunit bands of ASSAM separated by alkaline-urea polyacrylamide gel electrophoresis and that migrated to the same positions as apoSASSAM-1 and apoSASSAM-2 were labeled by anti-apoSASSAM-1 antiserum. The amino acid compositions of apoSASSAM-1 and apoSASSAM-2 were much the same and closely resembled those of ASSAM and mouse apoA-II. Sequence analysis of apoSASSAM and ASSAM revealed a blocked amino terminus. ApoSASSAM is considered to be a mouse apoA-II and probably transforms to amyloid fibril "ASSAM" in tissues through a process yet to be clarified.     

Phenazine methosulfate stimulation of ouabain-sensitive Rb+ uptake by HeLa cells: effects of respiratory inhibitors, anaerobiosis, and ascorbate

phenazine methosulfate (PMS) stimulates ouabain-sensitive Rb+ uptake by HeLa cells. This stimulation cannot be attributed to the effect of the dye on the intracellular Na+ or ATP content. Respiratory inhibitors, such as 5 mM NaCN and 5 microM rotenone, and anaerobic conditions enhance the stimulation of Rb+ uptake by PMS. Cellular respiration is stimulated, but lactate production is reduced in the presence of PMS, irrespective of the presence of respiratory inhibitors. Cellular NADH is oxidized markedly on addition of PMS plus inhibitors, but it is not affected by addition of the inhibitors only. In the presence of a high concentration of PMS, PMS-stimulated ouabain-sensitive Rb+ uptake is inhibited by addition of ascorbate. From these results it is concluded that Na+K-pump activity is closely related to the cellular redox state.     

Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture     

Diurnal K+ and Anion Transport in Phaseolus Pulvinus

Diurnal movement of Phaseolus leaf is caused by deformationof the laminar pulvinus located at the joint of the leaf bladeand the petiole. The plants were cultured in solutions withvarious ion compositions, and changes of K⁺, Na⁺, Ca²⁺, Mg²⁺,Cl^–, NO₃– and P₁ concentrations both in the upperand lower parts of the laminar pulvinus were measured. Culturein 10 mM KCl solution caused an increase in K⁺ and Cl^–concentrations both in the upper and lower parts without anysignificant change in the concentration of NO₃^–; culturein 10 mM KNO₃ solution caused an increase in K⁺ and NO₃^–concentration without any significant change in the concentrationof Cl^–; and culture in 10 mM KH₂PO₄ solution caused anincrease in K⁺ and P₁ concentrations without any significantchange in the concentrations of NO₃- and Cl^–. K⁺ moved from the upper to lower parts or from the lower toupper parts diurnally in all plants cultured in any solutionmentioned above. The main inorganic anion that accompanied thisK⁺ movement was Cl^– in KCl solution, and NO₃^– inKNO₃ solution. When the seedlings were cultured in distilledwater or in KH₂PO₄ solution, neither Cl^– NO₃^– norP₁ accompanied this K⁺ movement. In these cases, mainly H⁺ and/ororganic anions are supposed to move in exchange for and/or incombination with K⁺ movement. (Received November 8, 1982; Accepted June 13, 1983)