Two-dimensional gel analysis of zymogen-activating factors in the small intestine of the mouse

Zymogen-activating factors in the mouse were investigated by two-dimensional electrophoresis. Mouse pancreatic zymogens--trypsinogen-I group (Try G-I group), trypsinogen-II (Try G-II), and chymotrypsinogen (Chy G)--were purified using DEAE-cellulose column chromatography. Analysis by two-dimensional electrophoresis, using the purified zymogens as substrates, revealed enterokinase isozymes and chymotrypsinogen-activating factors in both the intestinal extract and luminal fluid. Mouse enterokinase was separated into at least two bands in the first-dimensional gel, each able to activate both trypsinogens Try G-I group and Try G-II. Chymotrypsinogen-activating factors were separated into several bands in the first-dimensional gel. Some activating factors showed mobilities similar to those of mouse enterokinase isozymes. Moreover, other activating factors that can activate chymotrypsinogen were present only in the more anodal area of the first-dimensional gel. These findings indicate that at least two enterokinases and several chymotrypsinogen-activating factors play an important role in the process of activating digestive enzymes.     

Wavelength-dependence of the relative rate constants for the main geometric and structural photoisomerization of bilirubin IX alpha bound to human serum albumin. Demonstration of green light at 510 nm as the most effective wavelength in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha via (EZ)-bilirubin.

The kinetics for the quantitatively important reaction: (Formula: see text) that is, the photochemical interconversion between bilirubin and its geometric and structural photoisomers bound to human serum albumin in aqueous solution when various wavelengths of monochromatic light were used, were assayed by h.p.l.c. In order to clarify the wavelength-dependence of the relative rate constants in the individual steps, a light-source with a half-bandwidth of 10 nm was used at increments of 20 nm, in the range from 410 nm to 550 nm. We describe for the first time studies on the wavelength-dependence of rate constants in geometric and structural photoisomerization reactions in vitro of (ZZ)-bilirubin or (EZ)-bilirubin bound to human serum albumin, especially the relative rate constants of cyclization of (EZ)-bilirubin into (EZ)-cyclobilirubin. Because studies in vitro have demonstrated that the wavelengths from 350 to 450 nm are mutagenic, the results obtained indicated that the safest and ideal light-source for phototherapy is green light of 510 nm, which keeps (ZE)-bilirubin concentrations as low as possible, as shown by a maximal value of k2 at 510 nm and a relatively low value of k1 at 510 nm. This light-source still ensures the substantial absorption of (ZZ)-bilirubin, which is the precursor of (EZ)-bilirubin, the intermediate in (EZ)-cyclobilirubin formation and, furthermore, as shown by the maximal value of k5 and a considerable value of k4 at 510 nm, promotes the cyclization of (EZ)-bilirubin derived from (ZZ)-bilirubin even though k3 at 510 nm also shows a peak value.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Immunotherapy with bestatin for acute nonlymphocytic leukemia in adults

Summary In a cooperative randomized control study of immunotherapy with bestatin in combination with chemotherapy in adults with acute nonlymphocytic leukemia (ANLL), 101 patients (48 in the bestatin group and 53 in the control group) out of 115 patients registered were evaluated as eligible. The bestatin group achieved a statistically significant prolongation of survival compared with the control group in overall ANLL and acute myelogenous leukemia. In the analysis of patient age, the bestatin group achieved a statistically significant prolongation of both the remission duration and survival in patients aged 50 to 65 years, while the differences were not significant in the 15 to 49 age group. The bestatin group tended to achieve a higher rate of reinduction of remission in patients who had recurrence of leukemia. Side effects developed in only 5 (9.6%) of 52 patients treated with bestatin. None of these side effects were particularly serious in nature. It is concluded that bestatin is useful for prolongation of survival of adult patients with ANLL, making for a longer remission duration especially in elderly patients and with few side effects.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Electron microscopy of two types of gonadotrophs in the anterior pituitary glands of persistent estrous and diestrous rats

Summary Persistent estrus and diestrus was produced in rats by the administration of estrone for either 5 days or 30 days, respectively, immediately after birth. Female rats without any treatment were used for control. After these rats grew up, the anterior pituitaries were examined by electron microscopy. The identification criteria for two types of gonadotrophs, FSH-and LH-cells, proposed by Barnes were adopted. In the persistent estrous rats, FSH-gonadotrophs were almost normal, but LH-gonadotrophs were filled with an abundance of secretory granules which were probably suppressed in discharge. On the other hand, in the persistent diestrous rats, FSH-cells were few in number and strongly atrophic, containing a few secretory granules, while LH-cells were almost normal or rather slightly activated. These electron microscopic findings well coincide with the results of light microscopy of ovaries, which suggested that in the persistent estrous rats FSH secretion might be almost normal but the secretion of LH might be inhibited, while in the persistent diestrous rats FSH secretion might be almost totally abolished but LH might be moderately secreted. From these findings, identification of FSH-and LH-gonadotrophs in the anterior pituitary of the rat well coincides with that proposed by Barnes in mice.     

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1^a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T^– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T⁺ chimeras). When lethally irradiated AKR (Mls-1^a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T⁺ and T^– chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T⁺ chimeras but not in T^– chimeras. On the other hand, clonal elimination of Vβ6⁺ T cells reactive to the recipient Ag (Mls-1^a) was abolished in T⁺ chimeras but successfully induced in T^– chimeras. The Vβ6⁺ T cells not eliminated in T⁺ chimeras showed depressed responses against Mls-1^a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.     

A rat cerebellar protein containing the cdc10/SWI6 motif.

Systematic analysis of soluble proteins in developing rat cerebellum by an automated two-dimensional liquid-chromatography system detected a number of proteins which increased transiently during the initial stage of postnatal development. One of the proteins, V-1, was isolated using a liquid-chromatography system, and its amino acid sequence was determined by analysis of the purified protein. The sequence showed that the V-1 protein consists of 117 amino acids with an acetylated N-terminus, and has 2.5 internal sequence repeats of 33 amino acids. Computer retrieval of the sequence indicated that the repeated sequences have a structural characteristics of the cdc10/SWI6 motif, which is found in a series of proteins, including those involved in cell-cycle control and cell-fate determination in yeast, Drosophila melanogaster and Caenorhabditis elegans. The structure of V-1, coupled with its controlled expression in early postnatal development, implies a potential role for V-1 in cerebellar morphogenesis.     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)