Macrophages containing langerhans cell granules in normal lymph nodes of the rabbit

Summary Light and electron microscopic studies on macrophages of normal rabbit lymph nodes showed two types, one with little phagocytic activity and many features similar to those of epidermal Langerhans cells. Among these are characteristic Langerhans cell granules. From these findings it is concluded that the Langerhans cells may be derived from lymph node macrophages.The helpful advice and criticism of Dr. Toshio Nagano, Department of Anatomy, are gratefully acknowledged. The discussion with Dr. Mitsumasa Itoh, Department of Dermatology, was also helpful.     

Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.

The borate transporter BOR1 senses the boron concentration through its borate transport activity for K63-linked polyubiquitination and subsequent degradation.     

Electrophysiological and histological study of synaptic connections between lateral geniculate transplant and host visual cortex

The lateral geniculate nucleus (LGN) of fetal Wistar rats was transplanted to the visual cortex (VC) of 33 neonatal Wistar rats. Histological examination showed transplanted cells in all the host brains. Intensively labeled cells were demonstrated in the transplant by labeling with true blue. Electrophysiological studies with brain slice preparations demonstrated that the transplanted LGN sent axons and made excitatory monosynaptic connections mainly in layer IV of the VC area 17. Corticogeniculate projections were also demonstrated in the transplanted LGN.     

Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs

Summary 67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and-4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.     

Countercurrent chromatography with a new horizontal flow-through coil planet centrifuge

A versatile horizontal flow-through coil planet centrifuge has been introduced for performing countercurrent chromatography. The apparatus carries a pair of coiled separation columns. Both of these allow continuous elution simultaneously without the use of rotating seals. One column enables preparative-scale separations and the other, analytical-scale separations, both with a high partition efficiency comparable to that obtained in liquid chromatography but without the complications arising from the use of solid supports. Capability of the apparatus was demonstrated on separation of dinitrophenyl amino acids using a two-phase solvent system composed of CHCl₃/CH₃COOH/0.1 HCl at a 2/2/1 volume ratio.     

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.     

Centrifugal precipitation chromatography

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.     

Primary malignant lymphoma of the uterine corpus and cervix. Report of a case with immunocytochemical analysis

In a 69-year-old woman, a gynecologic smear was the first indication of the presence of a nonepithelial malignant tumor. While first thought to represent an adenocarcinoma, malignant lymphoma was later cytologically suspected because of the presence of isolated large malignant cells with macronucleoli. The initial clinical and histologic studies failed to indicate a malignancy. The malignant cells in postoperative tissue samples showed a positive immunohistochemical reaction for leukocyte-common antigen (LCA) and a negative reaction for epithelial membrane antigen, confirming the cytologic suggestion of a uterine lymphoma. Immunocytochemical staining subsequently performed on the destained cytologic specimen gave a positive immunoreactivity to LCA in the cytoplasm of the malignant cells.