Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.     

Chest-wall reconstruction by contralateral latissimus dorsi musculocutaneous flap

Reconstruction of chest wall and axilla are performed in 11 patients using a contralateral latissimus dorsi musculocutaneous flap. The entire lattisimus dorsi muscle, including the fascial portion, safely carried an island of skin from the area of the lumbodorsal fascia to the contralateral axilla. The flap was transposed to the defect through a tunnel between the pectoralis major and minor muscles. Most patients who needed reconstruction of the chest wall and axilla had compromised ipsilateral vasculature that prohibited its use in a pedicled flap but had an intact contralateral chest wall, axilla, and thoracodorsal vessels. Therefore, this procedure was performed easily in comparison with a free flap or pedicled omental flap. This is a new, valuable application for the versatile latissimus dorsi musculocutaneous flap.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

The parasitoidOoencyrtus nezarae (Hymenoptera: Encyrtidae) prefers hosts parasitized by conspecifics over unparasitized hosts

Summary Two laboratory experiments were conducted to examine the ovipositional preferences of the egg parasitoidOoencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) for parasitized and unparasitizedMegacopta punctatissimum Montandon (Hemiptera: Plataspidae). Females that had never oviposited or that had not oviposite for 3 days preferred recently parasitized hosts more than unparasitized hosts. The preference for recently parasitized hosts appeared to be mediated by the punctures in already parasitized hosts made by the ovipositor of the first female. Survival of the parasitoid progeny was lower in recently parasitized hosts than in unparasitized hosts. However, handling time of parasitized hosts was extremely short relative to that of unparasitized hosts, because the superparasitizing female could use the punctures made by the previous females. It is concluded that the females preferred the parasitized hosts over unparasitized hosts because the benefit of saving time and energy for drilling was more than the cost of progeny survival.     

Lid margin reconstruction with an orbicularis oculi musculocutaneous advancement flap and a conchal cartilage graft

A simple method to reconstruct the midlateral lid margin defect is described using an orbicularis oculi musculocutaneous advancement flap and a free conchal cartilage graft. This method is easy to perform not only in the lower eyelid, but also in the upper one, provides a natural gray line and a stable lid margin without postoperative eversion, and substitutes for the Leone and van Gemert procedure.     

Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis.

BmFTZ-F1 is a Bombyx mori homologue of FTZ-F1, a positive regulator of the fushi tarazu gene of Drosophila melanogaster. In order to determine the sequence recognized with this factor, we made three sets of oligonucleotide mixture which contain 4 possible nucleotides at different positions within the previously proposed 12-bp binding consensus sequence. Oligonucleotides which bound to purified BmFTZ-F1 were separated by a gel mobility shift procedure and a binding sequence was determined by direct sequencing through Maxam-Gilbert method. By this analysis, 7 positions showed clear sequence preference and 5 positions showed weak or no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition analysis and results were presented as a quantitative difference in the binding affinity. From these analyses, we conclude that the best binding sequence of BmFTZ-F1 is 5'-PyCAAGGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.     

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.     

Immunocytochemical localization of 2,4-dienoyl-CoA reductase in the liver of normal and di-(2-ethylhexyl)phthalate-fed rats

Summary Localization of 2,4-dienoyl-CoA reductase (DCR) in rat liver was studied using immunoenzyme and immunogold techniques. The animals were fed on a laboratory diet with or without 2% di-(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for two weeks. For light microscopy (LM), semithin Epon sections were stained by immunoenzyme technique after removal of the epoxy resin. For electron microscopy (EM), ultrathin Lowicryl K4M sections were stained by the protein A-gold technique. By LM, in untreated rats reaction deposits showing the antigenic sites for DCR were present in the cytoplasmic granules. Hepatocytes, epithelial cells of interlobular bile duct, and sinus-lining cells contained these granules. After administration of DEHP, the cytoplasmic granules stained similarly. The staining intensity of the heaptocytes increased markedly, but that of the other cells decreased. The sinus-lining cells became mostly negative. By EM, gold particles indicating the antigenic sites for DCR were present in both the mitochondria and peroxisomes of hepatocytes of untreated rats. In the other cells, the gold label was confined to the mitochondria. After administration of DEHP, labelling intensity of the hepatocyte mitochondria increased markedly, but that of the peroxisomes conversely decreased. Quantitative analysis of labelling density showed that the mitochondrial DCR increased to about three times that in the untreated rat, but the peroxisomal DCR decreased to 1/6. The results show that in the rat liver, DCR exists in both, mitochondria and peroxisomes. DEHP can induce mitochondrial DCR, but not peroxisomal DCR.