Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.     

Immunoreactive dog C-peptide level in the pancreatic vein

C-peptide immunoreactivity (CPR) levels were measured in dog superior pancreaticoduodenal vein using synthetic dog C-peptide and its antiserum. The basal CPR level was approximately twice as high as the basal immunoreactive insulin (IRI) level on a molar basis. Glucose (10 mg/kg/min) or arginine (250 mg/kg/min) infusion for 5 min into the superior pancreaticoduodenal artery caused a prompt, parallel increase in IRI and CPR. IRI and CPR were closely equimolar at peak secretions. One bolus administration of synthetic neurotensin (10 microgram/kg) into the same artery produced a mild hyperglycemic response and biphasic IRI and CPR responses at 30 min in the vein. The IRI and CPR increases were closely equimolar during the first phase of secretion, but during the second peak a larger increase was found in CPR than IRI. Upon infusion of synthetic substance P (50 ng/kg/min) for 30 min, IRI and CPR concentrations showed a parallel and closely equimolar fall. These results indicate that insulin and C-peptide were released from beta cells in equimolar concentrations.     

Morphological study on early ontogeny of theGinkgo leaf

The early ontogeny of theGinkgo leaf was studied by scanning electron microscope. The lamina of the leaf is derived from the protuberance of the adaxial side of a leaf buttress. The protuberance shows successive bifurcations. The second bifurcation occurs approximately at a right angle to the first. Thus, the abaxial surface of the leaf is derived from the outer surface of the protuberance and the adaxial is derived from the depressed inner surface of the protuberance formed by the first and second bifurcations. It is supposed that thick veins along both edges of the lamina are differentiated by an uneven dichotomous branching system of procambia caused by positional relationships: both edges of the lamina are derived from the parts of the protuberance just above a pair of procambia which come from the stem. On the shoot apex, there are a central cell group, which consists of only a few cells, and radially-arranged cell files. The leaf primordium is initiated from a sector area which consists of several files among radially-arranged cell files.     

Structure of the vegetative shoot apex ofCassiope lycopodioides

The structure of the vegetative shoot apex ofCassiope lycopodioides D. Don, which has a decussate leaf arrangement, was analyzed using trans- and longisections to generate a three-dimensional viewpoint. The apical dome of this species is relatively high from the middle to the maximal area phase of a plastochron. Therefore, the initial protrusion of a pair of leaf primordia occurs laterally on an apical dome conspicuously in contrast to the cases ofDaphne pseudo-mezereum andClethra barbinervis whose apices are nearly flat or slightly convex. The structure of the apex ofCassiope, however, may be understood with the concept of “apical sectors” on the same basis asDaphne andClethra (Hara, 1961, 1962, 1971a, b, c).     

A randomized trial of chemoimmunotherapy of acute nonlymphocytic leukemia in adults using a protein-bound polysaccharide preparation

Summary The effect of immunotherapy with a protein-bound polysaccharide preparation termed PSK on remission duration and survival of adults with acute nonlymphocytic leukemia (ANLL) was studied in a prospective randomized cooperative trial. After having achieved complete remission and receiving a consolidation therapy, 73 patients were randomized either to maintenance chemotherapy or to maintenance chemotherapy plus immunotherapy with PSK. Ultimately 36 patients in the chemotherapy group and 31 in the chemoimmunotherapy group were evaluable. Six months after the last entry, immunotherapy with PSK showed a borderline beneficial effect on remission duration (P=0.089) and on duration of survival (P=0.062). When the data were analyzed 12, 18, and 24 months after the last entry there were no significant differences in duration of remission and survival between the two groups. However, analysis of the data of patients who had maintained complete remission for more than 270 days revealed that immunotherapy had a suggestive beneficial effect (P=0.105), prolonging the 50% remission period by 418 days (885 vs 467 days). Thus, immunotherapy with PSK seems to be active in the treatment of adult ANLL when used for maintenance therapy in combination with chemotherapy, especially in patients with a good prognosis.     

Neuroeffector actions of prostaglandin D2 on isolated dog mesenteric arteries

Treatment with prostaglandin (PG) D₂ in concentrations (10⁻⁸ to 10⁻⁷ M) insufficient to alter the basal tone potentiated the contractile response of helical strips of dog mesenteric arteries to transmural electrical stimulation but did not influence the response to norepinephrine. The potentiating effect of PGD₂ was not prevented by treatment with diphloretin phosphate, a PG antagonist, whereas contractions of dog cerebral arteries induced by PGD₂ were suppressed. The ³H-overflow evoked by transmural stimulation in superfused mesenteric arterial strips previously soaked in ³H-norepinephrine containing media was significantly increased by PGD₂. It is concluded that PGD₂ increases the stimulation-evoked release of norepinephrine from adrenergic nerves innervating the arterial wall. PGD₂ appears to act differently on receptive sites responsible for increasing the release of norepinephrine and for producing arterial contraction.     

VIP-immunoreactivity in the skin of various mammals: immunohistochemical, radioimmunological and experimental evidence for a dual localization in cutaneous nerves and merkel cells

In the present study VIP-immunoreactive (IR) nerve fibers were found in the skin of several mammalian species (cat, dog, pig and man). They supplied predominantly the arteries and arterial portions of arteriovenous anastomoses. Far fewer VIP-IR nerve fibers innervated veins and arterioles. Capillaries were supplied by VIP-IR fibers only in sweat and Meibomian glands. Some non-vascular VIP-IR nerve fibers were seen in contact with dermal smooth muscle strands. In eccrine sweat glands and in Meibomian glands VIP-IR fibers were targeting glandular cells. In addition, VIP-IR nerve fibers innervated the upper parts of facial hair follicles. In non-neuronal localization VIP-IR occurred in Merkel cells in all species and sites, while the intraepidermal axons consistently were not VIP-IR. Radioimmunoassay of different skin regions of cats also suggested both a neuronal and a Merkel cell origin of VIP-IR. Under physiological conditions VIP which is released from its neuronal and non-neuronal cutaneous pools may have an impact on thermoregulation by influencing blood flow and sweat production. It may also modulate axon-endings in Merkel cell-axon complexes and hair follicle receptors. Under pathological conditions an enhanced release of cutaneous VIP may lead to local inflammatory processes partly mediated via release of histamine from cutaneous mast cells.     

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.     

Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans

Summary Two PstI fragments (5.3x10⁶ and 4.3x10⁶ daltons) coding for Anacystis nidulans rRNA genes were cloned. The cloned rDNAs were characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis and the R-loop technique. The results indicated that both fragments contained 16S, 23S and 5S rRNA genes in this order. A tRNA gene(s) was detected in the spacer region between 16S and 23S rRNA genes. The organization of A. nidulans rRNA genes resembles those of E. coli and of Euglena chloroplasts rather than those of higher plant chloroplasts.