Characterization of beta-galactosidase from a special strain of Aspergillus oryzae.

beta-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from alpha-galactosidase, alpha- and beta-mannosidase, alpha- and beta-N-acetylhexosaminidase, and protease activities. The beta-galactosidase was capable of acting on aryl beta-galactosides, lactose, and lactosides. It also hydrolyzed beta-galactosyl linkages in urinary glycoasparagines and asialo alpha1-acid glycoprotein. The enzyme was rather stable in aqueous solution, retaining full activity at 4 degrees for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37 degrees, full activity was maintained for several days.

Structure of two glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU).

The structures of two glycoasparagines composed of one mole each of N-acetylneuraminic acid, galactose, N-acetylglucosamine, and asparagine were determined by periodate oxidation, enzymatic degradation, and methylation analysis. The structures were NANAalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta leads to Asn and NANAalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta leads to Asn, respectively.

Improvement in esophageal varices and liver histology postoperatively in Budd-Chiari syndrome.

BACKGROUND: In the past 17 years, 32 patients with Budd-Chiari syndrome were treated by reconstruction of the occluded inferior vena cava and reopening of the hepatic veins under femoro-femoral normothermic extracorporeal partial bypass. The mean follow-up was 8 years (range, 1.5 to 17 years). METHODS: To evaluate the benefits of our operative procedure, we compared the preoperative, early postoperative, and late postoperative endoscopic appearance of the esophageal varices and the histologic findings of the liver tissue obtained intraoperatively and at a later date. RESULTS: The esophageal varices found preoperatively in 29 patients (90.6%) had disappeared in 7 patients by the time of discharge, and in 2 patients they disappeared 4 to 7 years after surgery. In the remaining 20 patients, the grade of the esophageal varices was reduced markedly. Histologic examination of the liver showed cirrhosis in 22 patients, fibrosis in 9 patients, and severe congestion in 1 patient. Inspection of the liver in the late postoperative period (in 10 patients) showed improvement in centrilobular congestion and no increase in interlobular fibrosis. CONCLUSIONS: Gradual and steady improvement of esophageal varices and hepatic fibrosis can be achieved after our operative procedure.

Urinary excretion of a large amount of bound sialic acid and of under sulfated chondroitin sulfate A by patients with the Lowe syndrome.

Urine samples from two patients with the Lowe syndrome were analyzed for sialic acid and mucopolysaccharides. The sialic acid content, relative to the creatinine content, was 4--5 times higher in these patients' urine than in normal urine. Most of the sialic acid was found in unidentified glycoproteins of high molecular weight, but the levels of sialyllactose and free sialic acid were also elevated about 2 fold. A most remarkable finding was the excretion of undersulfated chrondroitin sulfate A without other mucopolysaccharides normally occurring in urine. It is suggested that a disorder in sulfation of mucopolysaccharides is etiologically implicated in the Lowe syndrome.

Left ventricular pseudoaneurysm after sutureless repair of subacute left ventricular free wall rupture: a case report.

A 65-year-old woman presenting with a left ventricular pseudoaneurysm 27 months after sutureless repair of a subacute left ventricular free wall rupture complicating acute myocardial infarction is described. An autologous pericardial patch and gelatin resorcin formaldehyde (GRF) glue were used in the repair. A small pseudoaneurysm bulged out over the true aneurysm of the left ventricle. We performed a Dor operation and concomitant bypass grafting to the right coronary artery. Although sutureless repair is an effective procedure for subacute left ventricular free wall rupture, left ventricular pseudoaneurysm in the late postoperative period may be a rare problem after this repair.

Cardiac reoperation in a patient with osteogenesis imperfecta: a case report.

A case of a 40-year-old man with dehiscence of the prosthetic aortic valve and recurrence of mycotic aneurysm of the left ventricular outflow tract with osteogenesis imperfecta is presented. He had an operation of aortic valve replacement and direct closure of the mycotic aneurysm for infective endocarditis twenty-one months ago. We performed reoperation of prosthetic aortic valve, patch closure of the mycotic aneurysm and graft replacement of the ascending aorta. He was complicated with multiple fractures of bilateral scapla and dislocation of left shoulder one postoperative day. Fortunately, cardiac reoperation was performed successfully in this patient despite anticipated difficulties with tissue friability with osteogenesis imperfecta.

Sutureless technique for subacute left ventricular free wall rupture: A case report of an 85-year-old.

This case was an 85-year-old female who developed left ventricular free wall rupture (LVFWR) of the anterior wall 13 days after an acute myocardial infarction. She was further complicated with an ascending aortic aneurysm and severe aortic regurgitation. The wall was repaired using a sutureless technique with an autologous pericardial patch and GRF glue without cardiopulmonary bypass. Although the complication of a left ventricular aneurysm was seen, the postoperative course was uneventful. Nevertheless, she is doing well 9 months after surgery.

Surgical treatment of multiple inflammatory aortic aneurysms of the aortic arch and thoracoabdominal aorta.

A 67-year-old woman hospitalized with pleuritis was treated with antibiotics. Although the inflammation was resolved, saccular aneurysms in the aortic arch and thoracoabdominal aorta enlarged rapidly. We conducted graft replacement of the aortic arch, but despite careful blood pressure control, the thoracoabdominal aneurysm rapidly enlarged even further. We conducted graft replacement of the thoracoabdominal aorta on day 25 after the first operation. The postoperative course was uneventful and no exacerbation was found 18 months after the second operation. These multiple aortic aneurysms were diagnosed as inflammatory because bacterial tests of blood and aneurysmal walls were all negative and cells infiltrating aneurysmal walls were pathologically plasma cells.

Acute aortic dissection associated with Sheehan's syndrome.

A 69-year-old woman with Sheehan's syndrome who suffered acute Stanford type A aortic dissection had received corticosteroids and thyroid hormones for over 20 years. The entire ascending aorta was replaced in emergency graft replacement. We administered twice the usual dose of methylprednisolone during cardiopulmonary bypass and twice the patient's usual dose of prednisolone from postoperative day 1 to 6. The usual 100 micrograms of levothyroxine sodium was given orally from postoperative day 1. The patient's postoperative course was uneventful. This case emphasizes the importance of early active supplementary treatment with steroids and thyroid hormones for major surgery in patients with Sheehan's syndrome.

Successful surgical treatment of impending rupture of thoracoabdominal aortic aneurysm in an elderly patient with severe pulmonary emphysema.

In a case of successful surgery for impending thoracoabdominal aortic aneurysmic rupture, an 83-year-old man with severe pulmonary emphysema was transferred to our hospital diagnosed with impending aneurysmic rupture. The aneurysm had been pointed out 2.5 years ago but surgical repair was not undertaken due to the patient's severe pulmonary emphysema. After admission, computed tomography showed an enlarging saccular thoracoabdominal aortic aneurysm. Emergency surgery was conducted because of severe pain below the left costal margin. We resected the wall of the saccular aortic aneurysm and reconstructed the aorta with an on-lay patch under femoro-femoral bypass and selective visceral organ perfusion. Tracheostomy provided respiratory care on the day following surgery. The patient was weaned from respiratory support 6 days after surgery. Postoperative aortography showed that the reconstructed thoracoabdominal aorta functioned satisfactorily. The patient remains in good health 18 months after surgery.

Surgical treatment of traumatic aneurysm of the ascending aorta.

Traumatic aneurysm of the ascending aorta is a rare event. This case describes a patient with such an aneurysm, resulting from injuries received in a motorcycle accident. The patient was admitted to the emergency room of a local hospital complaining of chest pain, and was subsequently referred to our institution. On admission, a chest x-ray showed mediastinal widening. Computed tomography and aortography revealed an ascending aortic aneurysm and contusion of the upper lobe of the right lung. Due to concerns about bleeding from the lung contusion, surgery was delayed for one week. During surgery, intimal tears were detected at two sites in the ascending aorta. The wall of the ascending aorta was subsequently resected and a prosthetic graft inserted. The postoperative period was uneventful and a postoperative aortogram showed that the graft had molded well.

Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy.

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO(2) values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology. Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll(®) gradient method and cultured on Matrigel(®) to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h. Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen. These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.

Activation of immune responses by muroctasin.

N2-[(N-Acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin), a muramyl dipeptide derivative, has been reported to enhance host resistance to infections, as well as displaying adjuvant activities on the humoral and cellular immune responses. This paper reviews briefly its immunopotentiating activities, which were found to be attributed to its augmenting effect on interleukin 1 production.

Effects of cyclophosphamide treatment and gamma irradiation of SJL mice on the IgE antibody response and the nature of IgE-binding factors.

Immunization with alum-absorbed ovalbumin (OA) failed to induce IgE antibody responses in SJL mice, while mice pretreated with either cyclophosphamide (CY) or gamma-irradiation prior to immunization transiently formed IgE antibodies. Spleen cells of OA-primed SJL mice formed IgE-suppressive factors upon incubation with OA. In contrast, spleen cells of the CY-treated or irradiated mice formed IgE-potentiating factors. It was found that CY treatment diminished the formation of glycosylation inhibiting factor (GIF) and enhanced the formation of glycosylation enhancing factor (GEF). The results suggest that the enhancement of the IgE antibody response by CY treatment is due not only to elimination of suppressor T cells but also to the activation of GEF-forming lymphocytes.

Carrier-specific suppression of antibody responses by antigen-specific glycosylation-inhibiting factors.

We previously established an ovalbumin (OA)-specific T cell clone from spleen cells of BDF1 mice, which had been treated by i.v. injections of OA, and constructed antigen-specific T cell hybridomas from the T cell clone. One of the hybridomas constitutively released glycosylation-inhibiting factor (GIF) which lacked affinity for OA, and was called non-specific GIF. Incubation of the same hybridoma cells with OA-pulsed syngeneic macrophages or OA-pulsed B lymphoblastoid cells of BALB/c origin resulted in the formation of GIF molecules that had affinity for OA but not for bovine serum albumin or keyhole limpet hemocyanin. Both the OA-specific GIF and nonspecific GIF bound to monoclonal anti-lipocortin and possessed I-Jb determinants. The OA-specific GIF consisted of two species of molecules, of m.w. 80,000 and 30,000 to 40,000, respectively, whereas the nonspecific GIF from unstimulated cells had an m.w. of 15,000. Intravenous injections of OA-specific GIF or nonspecific GIF into BDF1 mice suppressed both the IgE and IgG1 anti-hapten antibody responses of the animals to dinitrophenyl derivatives of OA (DNP-OA), but OA-specific GIF was much more effective than nonspecific GIF in suppressing the antibody responses. When the same preparations of GIF were injected into DNP-KHL-primed mice, OA-specific GIF and nonspecific GIF were comparable in suppressing the anti-DNP antibody response. In contrast to the 40,000 m.w. species of OA-specific GIF, the 80,000 m.w. OA-specific GIF had carrier-specific suppressive effects. The similarities of antigen-specific GIF to antigen-specific TsF suggest that the phospholipase-inhibiting activity of the molecules may be involved in the immunosuppressive effects of some antigen-specific TsF.

Induction of colony-stimulating factor and stimulation of stem cell proliferation by injection of muroctasin.

Modulation of myelopoiesis by anomeric mixture of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutamyl]-N6-stearoyl-L-lysin e (MDP-Lys(L18), muroctasin), a muramyl dipeptide analog, was investigated in mice. When BDF1 mice were subcutaneously treated with a single dose of 100 micrograms of MDP-Lys(L18), an increase in the number of peripheral blood leukocytes, a rise in the serum levels of colony-stimulating factor (CSF), proliferation of multipotential stem cells in the bone marrow and the spleen, and an expansion of granulocyte-macrophage progenitors in the bone marrow were observed. These findings suggest that the increase in the number of peripheral blood leukocytes in BDF1 mice by MDP-Lys(L18) is due to CSF production and stimulation of stem cell proliferation.

Modulation of the biologic activities of IgE binding factor. VI. The activation of phospholipase by glycosylation enhancing factor.

Glycosylation enhancing factor (GEF) from rat T cells is a kallikrein-like enzyme and enhances the assembly of N-linked oligosaccharides to IgE binding factors during their biosynthesis, whereas another T cell factor, i.e., glycosylation inhibiting factor (GIF), is a fragment of phosphorylated lipomodulin (i.e., phospholipase inhibitor), which when dephosphorylated inhibits phospholipase and the glycosylation process. The two T cell factors compete with each other when they are added to normal mesenteric lymph node cells during the formation of IgE binding factors. The addition of GEF to T cell hybridoma 23A4 cell switches the cells from the formation of unglycosylated IgE binding factor to the formation of N-glycosylated IgE binding factor. However, GEF neither inactivated GIF nor inhibited the formation of GIF by the T cell hybridoma. Stimulation of the T cell hybridoma with either affinity-purified GEF or bradykinin resulted in the release of GIF from the cells. GIF released by GEF stimulation had a m.w. of approximately 15,000 and bound to monoclonal antibody against lipomodulin. GEF and bradykinin also induced normal mesenteric lymph node cells to release GIF. Incorporation of 14C-arachidonic acid into 23A4 cells, followed by stimulation of the cells with GEF, resulted in the release of 14C-arachidonate. The results suggest that lipomodulin, a phospholipase inhibitory protein, is present in lymphocytes, and indicate that GEF and bradykinin induce the activation of phospholipase by stimulating cells to release lipomodulin.