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Arterioportal fistula (APF) combined with a visceral artery pseudoaneurysm is an exceptionally rare and critical vascular disorder of the abdominal viscera, with pseudoaneurysm rupture being potentially fatal and severe APF leading to portal hypertension, both of which necessitate immediate intervention. An 87-year-old woman with a history of pancreatitis presented with upper abdomen and back pain. Laboratory tests revealed elevated amylase levels and severe anemia. A computed tomography (CT) scan showed a large dorsal pancreatic artery (DPA) pseudoaneurysm with a fistula to the main portal vein. Given her advanced age, surgery was deemed high-risk, and endovascular treatment was selected. Transcatheter arterial embolization was successfully performed using coils to embolize the DPA pseudoaneurysm. A follow-up CT 1 week postprocedure confirmed the absence of a pseudoaneurysm and no further progression of anemia., (© 2024 The Authors. Published by Elsevier Inc. on behalf of University of Washington.)

Background: At craniotomy, the dura shrinks due to the drying effect of illumination and air exposure, rendering its primary closure difficult. When the opening is narrow, it can be patched with autologous grafts. However, tissue harvesting and meticulous suturing take time. As the cost-effectiveness of using dural substitutes is poor, we developed a new technique that involves dural scratching for the primary closure of dural openings., Methods: We applied our technique because in 53 adults who underwent supratentorial craniotomy we encountered difficulties with the primary closure of a dural opening 5 mm or less in width. With a scalpel, we placed several fine scratches parallel to the edge of the opening on the surface of the dura, taking care not to perforate the deep layer. This relieved dural tension and the tissue was stretched enough to permit closing with sutures., Results: Our technique achieved primary dural closure in 46 of 53 patients (87%) who primarily had undergone narrow supratentorial craniotomies. The other 7 additionally required small autologous grafts to patch narrow residual openings at the intersection of the durotomy. There were no procedure-related complications such as cerebrospinal fluid leakage and persistent headache due to tension of the closed dura., Conclusions: Dural scratching is simple, safe, requires no special instrumentation, facilitates primary closure of the shrunken dura by stretching, and reduces the need for patch grafting., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Carotid endarterectomy (CEA) and carotid artery stenting (CAS) are common treatments for ischemic stroke prevention in patients with carotid artery stenosis. However, the beneficial effects of CEA/CAS for cerebral hypoperfusion due to carotid artery stenosis have yet to be fully established. As dizziness is a common symptom in patients with carotid artery stenosis, we aimed to evaluate the effects of CEA/CAS on cerebral function in patients with carotid artery stenosis, using equilibrium function tests., Methods: This prospective observational cohort study included 50 patients who had carotid artery stenosis and were scheduled to undergo CEA or CAS. Before CEA/CAS, we quantitatively evaluated the vestibulo-ocular reflex (VOR) and vestibular evoked myogenic potential (VEMP), as indicators of brainstem/inner ear functions related to balance, and visual suppression of VOR, as an indicator of cerebral control over the brainstem reflex related to balance. These were then compared with supratentorial cerebral blood flow (CBF). Changes in VOR, VEMP, visual suppression of VOR, CBF, and dizziness after CEA/CAS were also evaluated., Results: The visual suppression rates of VOR correlated with supratentorial CBFs before CEA/CAS (correlation coefficient = 0.47, p = 0.003). The visual suppression rates of VOR (p < 0.001) and supratentorial CBFs (p = 0.003) were improved by CEA/CAS, while VOR and VEMP did not change. Symptoms of dizziness improved after CEA/CAS., Conclusions: Our results suggest that visual suppression of the VOR may be a novel and practical marker for the beneficial effects of CEA/CAS on supratentorial cerebral function., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Preoperative identification of the carotid bifurcation (CB) location and plaque and stenosis distal end of the cervical internal carotid artery in relation to bony structures is essential for carotid endarterectomy (CEA). However, for patients with contrast contraindications, cervical 3-dimensional computed tomography angiography (3D-CTA) is unavailable. In this study, we created fusion images of magnetic resonance angiography (MRA), black-blood (BB) magnetic resonance imaging (MRI), and CT to determine if these noncontrast agent images are superior to 3D-CTA for preoperative CEA planning., Methods: The fusion images showed vascular structures obtained by MRA, plaque observed by BB-MRI, and bone structures shown by CT. Spatial localization accuracy was verified by directly overlaying contrast-enhanced 3D-CTA images on the fusion images. We validated this technique in 50 patients with unilateral ICA stenosis, 28 of whom underwent CEA. The 2D-distance CB MRA-CTA (the 2D distance difference between CB MRA and CB CTA perpendicular to the long axis of the carotid artery) was measured. We also compared the findings of the fusion image regarding the CB location and plaque distal end with the operative findings., Results: The median 2D distance CB MRA-CTA was 1 mm. CB MRA was located distal and proximal to CB CTA in 21and 29 patients, respectively. The CB location and fusion-image plaque were consistent with the intraoperative findings in all CEA patients., Conclusions: Fusion images created from MRA, BB-MRI, and noncontrast CT were feasible as an alternative to 3D-CTA for patients with contrast contraindications., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Despite holding soft tissue away from high-speed drills during surgery, tissue can unexpectedly wrap around the drill shaft. We performed experiments to examine what precipitates such mishaps., Methods: In a windless environment, a Signature Drill System (Stryker) featuring a 4-mm diameter coarse diamond or cutting bit was placed parallel to a suspended strip of polypropylene. The distance between the end of the strip and the drill shaft or bits was 4 or 8 mm. In another experiment, we placed the drill horizontally 10 mm above the top of dry-ice fog. The maximum drill speed was 75,000 rpm, and the horizontal motion of the polypropylene strip and the vertical motion of the dry-ice fog were recorded by a video camera., Results: In the experiments, the strip parallel to the vertical shaft and the dry-ice fog were pulled toward the shaft; the higher the revolving drill speed, the faster its motion. On the other hand, in experiments where the end of the strip was next to either bit, no such motion was observed., Conclusions: The pulling force generated by revolving high-speed drill shafts may result in tissue wraparound even when soft tissue is held away from the shaft., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Chronic subdural hematomas (CSDHs) with narrow or multilayered hematomas must be accurately localized for burr hole drainage. We present a simple alternative localization method using conventional computed tomography (CT) scans acquired for the initial diagnosis and a commercially available carpenter's square (CS)., Methods: Using our novel method, we localized 77 narrow or multilayered CSDHs. A single burr hole site on the thickest portion of narrow hematomas or on the site allowing access to both the isolated superficial layer and the deep layer of multilayered hematomas was identified on axial CT images; the image was parallel to the orbitomeatal line (OML). On the target CT slice, the level from the OML and from the surface of the forehead to the intended burr hole (sagittal distance) was measured. The OML and the level of the CT target slice were marked using the CS. Then, the CS was placed at the marked target level; the 2 edges of the CS were situated on the frontal and temporal scalp and parallel to the OML and the sagittal line. The sagittal distance was then marked., Results: All burr holes reached the CSDHs. The mean deviation of the burr holes was 4.7 mm inferior and 1.4 mm anterior to the intended site. In 65 instances (84.4%), a deviation within 10 mm was observed in both the superoinferior and the anteroposterior directions., Conclusions: Our simple and inexpensive method can localize narrow or multilayered CSDHs with acceptable accuracy and increases the efficiency of routine clinical work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Many health care workers around the world tackled with COVID-19, however sadly, the infection of many medical care workers were reported. To reduce the risk of infection, we launched selected team (Team COVID) of non-specialists and brought in active telemedicine method and computed tomography (CT)-first protocol. We describe our actual practice and the health status of medical doctors dealing with COVID-19 patients., Methods: Between April 17, 2020 and May 24, 2020, 10 doctors worked with COVID-19 patients as part of Team COVID. The Team COVID doctors used a CT-first triage protocol for outpatients and telemedicine for inpatients and outpatients. We evaluated paired serum-specific antibodies for SARS-CoV-2 at the initial and end of the study duration and PCR results for SARS-CoV-2 at the end of the study duration. Furthermore, 36-item short-form of the Medical Outcome Study Questionnaire (SF-36) at the beginning and end of the study period were evaluated., Results: Ten doctors worked as Team COVID: seven internal medicine doctors and three surgeons. During the study period, Team COVID treated 165 individuals in the outpatient clinic and isolated hospitalized patients for 315 person-days. There were no positive results of serum-specific antibody testing and PCR testing for SARS-CoV-2 in Team COVID doctors. Furthermore, the SF-36 showed no deterioration in physical and mental QOL status. No in-hospital infection occurred during the study period., Conclusions: The Team COVID fulfilled the treatment using the active telemedicine and CT-first triage protocol without in hospital infection and excess stress. The combination strategy seems acceptable for both the protection and stress relief among the medical staff., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Ovarian cancer (OC) is an intractable gynecological tumor, and frequent recurrence is experienced within a few years even after the complete eradication of tumor tissues by radical resection and neo-adjuvant chemotherapies. The conventional recurrence marker, CA125, is widely used for follow-up after resection of OC, but CA125 has a long half-life in blood and lacks dynamic responses to tumor recurrence. Recent developments in liquid biopsy procedures are expected to overcome the difficulties in early diagnosis of OC recurrence after surgery., Methods: We applied droplet digital PCR (ddPCR) technology to detect circulating tumor-derived DNA in OC patients' plasma during follow-up. Exome sequencing of 11 tumor-normal pairs of genomic DNA from consecutive OC patients identified tumor-specific mutations, and ddPCR probes were selected for each sample., Results: Six of 11 cases showed apparent recurrence during follow-up (mean progression-free survival was 348.3 days) and all six cases were positive in ddPCR analyses. In addition, ddPCR became positive before increased plasma CA125 in five out of six cases. Increased allele frequency of circulating tumor DNA (ctDNA) is associated with increased tumor volume after recurrence. ddPCR detected ctDNA signals significantly earlier than increased CA125 in the detection of OC recurrence by imaging (49 days and 7 days before, respectively: p < 0.05). No ctDNA was detected in the plasma of recurrence-free cases., Conclusions: Our results demonstrate the potential of identifying ctDNA by ddPCR as an early detection tool for OC recurrence., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: The occipital transtentorial approach (OTA) is a very useful but challenging approach to expose the pineal region because the deep-seated arachnoid membranes usually fold and extend over the great vein of Galen (GVG), leading to dense and poor visibility. In addition, the practical aspects of arachnoid anatomy are not well understood. We aimed to develop a safe surgical procedure for the OTA according to the practical aspects of arachnoid anatomy., Methods: The procedure is shown through an illustrative video of surgery and cadaver. Five cadavers were analyzed for their arachnoid structures and the surgical procedures via the OTA, in strict compliance with legal and ethical requirements., Results: All cadavers showed a 2-layered arachnoid structure-one belonging to the occipital lobe, and the other to the cerebellum. According to our cadaveric analysis, the arachnoid attachment of the tentorial apex can be peeled bluntly, with an average distance of 10.2 mm. For our clinical presentation, a pineal tumor with hydrocephalus was detected in a 14-year-old boy. While using the OTA and expanding the deep surgical field, we detached the membrane from the tentorial apex and bluntly peeled it to reveal the deep veins. Finally, gross total removal of the tumor was achieved., Conclusions: A 2-layered arachnoid structure interposes the GVG from above and below the tentorium. The arachnoid membrane below the tentorium can be peeled off bluntly from the GVG to the attachment bundle limited by the penetrating veins. This detachment technique is useful for safe enlargement of the surgical field for the OTA., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Ovarian cancer is the most lethal gynecologic malignancy due to the tumor's acquisition of chemoresistance to platinum-based chemotherapy. To solve this problem, we conducted RNAi-based large-scale screening and determined that tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) is a key molecule involved in the platinum resistance of ovarian cancer cells. Recently, a variety of studies have investigated that small extracellular vesicles (sEVs) contribute to the communication between cancer cells, including the development of chemoresistance in ovarian cancer. The purpose of our study is to determine if sEVs-derived TIE-1 is involved in the chemoresistance of ovarian cancer cells., Materials and Methods: TIE-1-overexpressed TOV112D cells, termed TOV112D TIE-1 cells, were established, and sEVs were isolated from TOV112D TIE-1 cells supernatants by ultracentrifugation. We assessed cisplatin sensitivity in recipient cells with TOV112D TIE-1 -derived sEVs by cell-Titer Glo kit. We also asked whether sEV-derived TIE-1 suppressed the DNA damage response in recipient cells and evaluated the DNA damage response by counting cells positive for DNA damage foci., Results: TIE-1 was contained within sEV TIE-1 derived from the cellular supernatant of TOV112D TIE-1 . We showed that sEV-derived TIE-1 decreased chemosensitivity to cisplatin by suppressing the DNA damage response in recipient cells., Conclusion: Our findings suggest that sEV-derived TIE-1 could be a new therapeutic target for refractory ovarian cancer., (Copyright © 2021. Published by Elsevier Ltd.)

Objective: Cluster-like headache (CLH) associated with pituitary adenoma (PA) is rare. Although numerous cases have been reported, no summary of the literature has been published. Furthermore, the mechanism and efficacy of medication in CLH associated with PA remains unknown., Methods: We reviewed 14 cases of CLHs associated with PA published in the English and Japanese literature. We have also included and presented our experience with such a case., Results: The median age of patients with CLHs associated with PA was 46 years (range, 17-58 years). The ratio of men to women was 14:1. Headache duration ranged from 15-480 min, with left fronto-orbital pain being common. The most common autonomic nervous symptoms were eye-related in 13 patients (86.6 %), followed by nasal symptoms in 12 (80.0 %). Thirteen patients (86.6 %) had functional adenomas; the remaining two were nonfunctional. Twelve of the functional adenomas were lactotroph adenomas (80.0 %), and one was a somatotroph adenoma (6.6 %). CLHs significantly improved after cabergoline administration in 7/9 patients with a lactotroph adenoma (77.7 % response rate). In 5/11 patients with either a functional or nonfunctional PA who received a triptan, CLHs improved (45.4 % response rate)., Conclusion: Based on the efficacies of cabergoline and triptans, two different mechanisms may coexist in the pathogenesis of CLHs associated with PA: endocrinological and physical effects of the tumor itself. Cabergoline is the first-line treatment for headaches caused by lactotroph adenomas. Triptans can be effective as an acute drug for headaches associated with nonfunctional PAs and persistent headaches that remain after cabergoline administration., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Few papers have examined the association between the chemical components of PM 2.5 and health effects. The existence of an association is now under discussion., Methods: This case-crossover study aimed to examine the association between the chemical components of PM 2.5 and night-time primary care visits (PCVs) due to asthma attacks. The subjects were 1251 children aged 0-14 years who received medical care for asthma at a municipal emergency clinic. We measured daily average concentrations of hydrogen ion, sulfate ion, nitrate ion and water-soluble organic compounds (WSOCs), which are components of PM 2.5 . We estimated the odds ratios (ORs) of PCVs per unit increment (inter quartile ranges) in each chemical component of PM 2.5 for the subgroups of warmer months and colder months separately., Results: No association was seen between PCVs and PM 2.5 mass concentrations the day before the PCVs in either warmer or colder months. In the warmer months, an association was seen with the concentrations of WSOCs and hydrogen ion the day before the PCVs (OR = 1.33; 95% CI: 1.00-1.76, OR = 1.18; 95% CI: 1.02-1.36, respectively). Furthermore, a negative association was seen between sulfate ion and PCVs (OR = 0.85; 95%CI: 0.74-0.98). No associations were observed in the colder months., Conclusions: We observed a positive association between PCVs and certain concentrations of WSOCs and hydrogen ions in warmer months. In contrast, sulfate ion showed a negative association., (Copyright © 2019 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Leptomeningeal metastasis (LM) is rarely observed in gynecologic cancers. As gadolinium-enhanced magnetic resonance imaging (Gd-MRI) is highly effective for diagnosing LM, the aim of this study is to describe the clinical behaviors and outcomes of LM patients who were diagnosed by Gd-MRI. After securing institutional review board approvals, we retrospectively reviewed patient records. Eight patients were found to have LM from gynecological malignancies. Primary tumors included three ovarian cancers, one tubal cancer, one peritoneal cancer, two endometrial cancers, and one cervical cancer. Gd-MRI of the brain and the spine is indicated as the high-priority inspection for the diagnosis of this devastating complication., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Effects of an antiulcer drug, geranylgeranylaceton (GGA), and/or heat-stress on 72 kDa heat shock protein (HSP72) expression and protein content in cultured skeletal muscle cells were studied. Mouse skeletal muscle cells (C(2)C(12)) were subjected to either 1) control (cultured at 37 degrees C without GGA), 2) GGA administration (10(-11) - 10(-8) M), 3) heat-stress at 41 degrees C for 60 min, or 4) GGA administration combined with heat-stress. Expression of HSP72 was up-regulated by GGA administration. Heat-stress further enhanced the GGA-related up-regulation of HSP72. Administration of GGA caused an increase of muscular protein content as a dose-dependent manner. Protein synthesis was also stimulated by heat-stress alone in myotubes. It was suggested that GGA stimulates the differentiation of myoblasts and protein synthesis. These observations may also suggest that the administration of GGA could be one of the useful tools to gain muscular mass not only in athletes, but also in patients during rehabilitation.

Background: Skeletal muscle stem cells, so-called muscle satellite cells, are responsible for the repair and the regeneration of adult skeletal muscle tissues. Heat stress can facilitate the proliferation and the differentiation of myoblasts in vitro and can enhance their proliferative potential, which may stimulate the regrowth of atrophied skeletal muscle. The purpose of this study was to investigate the effect of heat stress on the regeneration of skeletal muscle injury induced by cardiotoxin., Methods: Male Wistar rats, aged 7 weeks, were randomly divided into six groups: a nonheated control group that received a physiological saline injection, a group heat stressed before physiological saline injection, a group heat stressed after physiological saline injection, a group injected with cardiotoxin without heat stress, a group heat stressed before cardiotoxin injection, and a group heat stressed after cardiotoxin injection (25 in each group). To initiate muscle injury and regeneration, 0.5 ml of 10 microM cardiotoxin was injected into the left tibialis anterior muscle. Conscious rats in some groups were exposed to environmental heat stress (41 degrees C for 60 min) in a heat chamber 24 h before or immediately after cardiotoxin or physiological saline injection. The heating protocol in the present study causes an increase in the colonic temperature to 41 degrees C. The left tibialis anterior muscles were dissected 1, 3, 7, 14, and 28 days after injection of cardiotoxin or physiological saline., Results: The wet weight and water content of muscles increased 1 day after cardiotoxin injection regardless of the application of heat stress, but normalized after 7-14 days. The muscle protein content in control rats had increased 7 days after heat stress. Although the muscle protein content decreased on cardiotoxin injection, heat stress caused a significant recovery in protein level. Expression of heat shock protein 72 (HSP72) and the number of Pax7-positive nuclei decreased after cardiotoxin injection but increased on the application of heat stress in both normal control and cardiotoxin-injected groups., Conclusions: Heat stress stimulated not only the proliferation of satellite cells but also protein synthesis during the regeneration of injured skeletal muscle. It is thus strongly suggested that the heating of injured skeletal muscle may facilitate recovery. There was no direct relationship between the level of HSP72 expression and muscle protein content, suggesting that HSP72 expression may not be the key signal for protein synthesis in the necrosis-regeneration process.

We obtained two mutant strains of respiratory syncytial virus (RSV) which showed resistance against NMSO3 after 15 and 33 passages, respectively, in HEp-2 cells in the presence of 6.8 microM of NMSO3. The EC50 values of NMSO3 for the resistant virus strains were 0.48 and 0.93 microM, that is 4.8-9.3 times higher than that of the parent strain (EC50 = 0.1 microM). The most resistant strain also showed resistance against heparin but was sensitive to dextran sulfate and a polyoxotungstate, PM-523. In order to determine whether the acquisition of resistance to NMSO3 was the result of the accumulation of genetic changes of virus, we sequenced the G- and F-protein genes. In comparison with the standard type of RSV strains, we identified changes of 10 amino acids in the G protein including those at the central conserved segment. However, we did not observe any particular changes in the amino acid sequence of the F-protein of the resistant strains. From these results, we conclude that NMSO3 inhibits the G-protein interaction to the receptor. The mutations in the G-protein may result in the observed phenotypic resistance of RSV towards NMSO3.

To determine the antiviral effects of compounds against ocular adenovirus (AdV) infection, we established an animal model of AdV infection in cotton rat eyes. Cotton rat eyes were inoculated intrastromally and topically with four AdV serotypes 4, 5, 8, and 37, and treated topically with 1% HPMPC (cidofovir) eye drops twice a day. The infected corneas were extracted and homogenized, and virus titers in the cornea specimens were determined by a plaque assay. The virus titer in AdV type 5-inoculated eyes peaked on days 0 through 3 after inoculation and virus shedding was detected for 18.0+/-2.8 days. AdV 5 antigen in the infected corneas was demonstrated in the corneal epithelial cells by immunofluorescence stain. However, for AdV serotypes 4, 8, and 37, no evidence of continued virus replication in cotton rat eyes was noted. Specimens from cidofovir-treated eyes infected with AdV 5 demonstrated a statistically significant reduction in the mean virus titer (days 3-15) (P=0.028) and virus shedding duration (P=0.0014), as compared with those of the control group.

The STAM family proteins, STAM1 and STAM2/EAST/Hbp, are phosphotyrosine proteins that contain SH3 domains and ubiquitin-interacting motifs. Their yeast homologue, Hse1, and its binding protein, Vps27, are involved in the vacuolar membrane transport machinery. Here we show that STAM1 and STAM2 are localized to the endosomal membrane. Some of these complexes contain Eps15, an endocytic protein, which accumulates in clumps upon expression of a dominant-negative form of Vps4-A, an AAA-type ATPase, that is required for normal endosome function. These results support the idea that the STAMs are mammalian vacuolar protein sorting (Vps) proteins. We also demonstrate that ligand-mediated epidermal growth factor receptor (EGFR) degradation is partially but not completely impaired in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) mouse embryonic fibroblasts. Furthermore, endosome swelling is seen in both Hrs(-/-) and STAM1(-/-)STAM2(-/-) cells. These results suggest that the STAMs and Hrs play important roles in the mammalian endosomal/vacuolar protein sorting pathway.

Seven polyoxotungstates substituted with vanadium or titanium atoms were examined for their activity against Flaviviridae (Dengue fever virus, DFV), Orthomyxoviridae (influenza virus type A, fluV-A), Paramyxoviridae (respiratory syncytial virus, RSV, parainfluenza virus type 2, PfluV-2 and canine distemper virus, CDV) and Lentiviridae (human immunodeficiency virus type 1, HIV-1) families. Among the seven polyoxotungstates examined, PM-43 [K(5)[SiVW(11)O(40)]], PM-47 [K(7)[BVW(11)O(40)]], and PM-1001 [K(10)Na(VO)(3)(SbW(9)O(33))(2)]26H(2)O contained vanadium. PM-1002 had the same core structure of (VO)(3)(SbW(9)O(33))(2) as PM-1001; however, three V atoms of PM-1001 consisted of two V(IV) and one V(V) and those of PM-1002 consisted of three V(IV). On the other hand, PM-518 [[Et(2)NH(2)](7)[PTi(2)W(10)O(40)]], PM-520 [Pri(2)NH(2)](5)[PTiW(11)O(40)] and PM-523 [PriNH(3)](6)H[PTi(2)W(10)O(38)(O(2))(2)]H(2)O all contained titanium. All compounds showed broad spectrum antiviral activity against all viruses examined except for PMs-43, -518 and -523 which did not exhibit inhibitory activity at >/=50 microM against PfluV-2, CDV and DFV, respectively. All compounds were inhibitory against HIV replication at an EC(50) of less than 2.0 microM. Among them, PMs-1001 and -1002 showed the most potent inhibition. The compounds were not toxic for MDCK, HEp-2 and Vero cells at a concentration of 200 microM. For the exponentially growing MT-4 cells, the vanadium containing polyoxometalates (PMs-43, 47, 1001, 1002) showed toxicity at concentrations between 41 and 47 microM. On the other hand, titanium containing polyoxometalates (PMs-518, -520, -523) were not toxic at 100 microM. The mechanism of anti-HIV action of PM-1001 was analyzed: it affected the binding of HIV to the cell membrane and syncytium formation between HIV-infected and uninfected cells.

NMSO3, a sulfated sialyl lipid, was evaluated for its efficacy against adenovirus (AdV) in vitro. The median effective concentration (50% effective concentration, EC(50)) of NMSO3 against replication of AdV type 2 (AdV2), type 4 (AdV4), type 8 (AdV8) and type 37 (AdV37) was 0.21-0.71 microg/ml in HEp-2 cells and 1.01-1.41 microg/ml in MKN-28 cells. The EC(50) values of NMSO3 were lower than those of HPMPC and ddC, which were also evaluated. NMSO3 exhibited minimal cytotoxicity against HEp-2 cells and MKN-28 cells, both for which the median cytotoxic concentration (50% cytotoxic concentration, CC(50)) was more than 1000 microg/ml. NMSO3 was the most potent and selective anti-AdV compound of those examined. NMSO3 inhibited AdV infection of HEp-2 cells only when present during the virus adsorption period. A virus binding assay using radiolabeled AdV4 revealed that NMSO3 inhibited viral binding to the HEp-2 cells. NMSO3 itself bound to the virus particles, but not to the HEp-2 cell membrane. Thus, the mechanism of anti-AdV activity by NMSO3 involves inhibition of virus adsorption to cells by NMSO3 binding to viral particles.

Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE) was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory components of SE were found to be heterogeneous anionic polysaccharides with different ion charges. The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the blockade of the virus attachment to cells.

The antiviral effects of a 20-mer antisense phosphorothioate oligonucleotide, PB2-as, on influenza A virus infection in mice were examined and compared to those of PB2-as encapsulated with several cationic liposomes. Intravenous injection of PB2-as, as a complex with DMRIE-C, a cationic liposome, was most effective for prolonging the mean survival time in days (MSDs) and increasing the survival rates of mice infected with the influenza A virus. In addition, the liposomal PB2-as significantly inhibited viral growth in lung tissues. These results suggest that PB2-as encapsulated with DMRIE-C may be active against the influenza A virus infection through the inhibition of virus replication in the mouse lung., (Copyright 2000 Academic Press.)

NMSO3, a sulfated sialyl lipid was evaluated for its efficacy against respiratory syncytial virus (RSV) and other myxovirus infections in cell culture. The median effective concentration (50% effective concentration, EC(50)) of NMSO3 against replication of the Long strain of RSV in HEp-2 cells was 0.2 and 0.32 microM by optical ELISA and the plaque reduction method, respectively. On the other hand, the corresponding values for ribavirin were 10.5 and 11.2 microM, respectively. NMSO3 showed potent activity against other laboratory strains as well as fresh clinical isolates of RSV, and the average EC(50) was similar to that for Long strain. NMSO3 exhibited minimal cytotoxicity against HEp-2, MDCK, HMV-2 and Vero cells for which the median cytotoxic concentration (CC(50)) was more than 685 microM. The selectivity index [SI=(CC(50) for HEp-2/EC(50))] of NMSO3 for RSV exceeded 2978 and that of ribavirin was 6. The EC(50) of NMSO3 against influenza virus (FluV) A (H3N2) was 23.8 by the MTT method using HMV-2 cells, and 17.8 microM by the TCID(50) method using MDCK cells. NMSO3 did not inhibit replication of influenza B virus, parainfluenza virus type 2 and canine distemper virus at 103 microM. NMSO3 inhibited RSV infection of HEp-2 cells when it was added between 0 and 1.5 h after virus infection. By a temperature shift experiment during the period of contact between the virus and cells, NMSO3 inhibited both the binding of RSV to the cells and its penetration into the cells. Prophylactic and therapeutic efficacy of NMSO3 against RSV infection in cotton rats was examined. Intraperitoneal administration of 100 mg/kg per day of NMSO3 to cotton rats from 1 day before or 1 h after to 3 days after the RSV infection, once a day every day, decreased the RSV titer in lungs to 10(-1.26) to 10(-1.63) compared to the control rats which were infected with RSV and left untreated.

In our extensive screening of anti-HIV-1 agents in chronically infected cell lines, we have found acridone derivatives to be selective inhibitors of HIV-1 replication. Among the acridone derivatives, 1-hydroxy-10-methyl-9,10-dihydroacrid-9-one (RD6-5071) suppressed tumor necrosis factor (TNF)-alpha-induced HIV-1 expression in the latently infected cell line OM-10.1, U1, and ACH-2. Its 50% effective concentration for HIV-1 p24 antigen production was 2.0 microg/ml in OM-10.1 cells, while its 50% cytotoxic concentration was 18 microg/ml. The compound also inhibited phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 expression in these cell lines. Furthermore, RD6-5071 was inhibitory to HIV-1 replication in acutely infected U937 and peripheral blood mononuclear cells. The compound was found to suppress TNF-alpha-induced HIV-1 long terminal repeat-driven gene expression. An inhibition assay for protein kinase C (PKC) revealed that RD6-5071 could reduce the enzyme activity. Furthermore, the compound was a moderate inhibitor of PMA-induced nuclear factor kappaB (NF-kappaB) activation, as determined by a gel mobility shift analysis. These results suggest that the acridone derivatives suppress HIV-1 replication at the transcriptional level primarily through a mechanism of PKC inhibition.

Mammalian cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) sequence informations. These fragments were subsequently used to screen a rat liver cDNA library, yielding a 1573-bp clone. Expression of DNA fragment containing either of two possible open reading frames (nucleotide numbers 97-1431 and 148-1431) of the isolated clone in yeast led to the accumulation of gamma-linolenic acid in the presence of exogenous linoleic acid. In this system, the addition of alpha-linolenic acid also resulted in the accumulation of its Delta-6 desaturated product whereas dihomo-gamma-linolenic acid failed to be a substrate. These results indicate that the protein encoded by the rat cDNA is Delta-6 fatty acid desaturase, and the first 17 amino acids corresponding to the coding region 97-147 of the clone are not required to function in yeast., (Copyright 1999 Academic Press.)

Benzodithiin derivatives are highly potent and specific inhibitors of respiratory syncytial virus (RSV) replication in vitro. The most potent and selective congener of a benzodithiin derivative is 1,4-dihydro-2,3-benzodithiin(RD3-0028). According to the modified 3-(4,5-dimethylthiazole-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay developed in our laboratories, this compound has a 50% effective concentration of 4.5 microM and a 50% cytotoxic concentration of 271.0 microM, which is superior to that of ribavirin. This compound also inhibits RSV strain subgroups A and B and clinical isolates. RD3-0028, however, does not inhibit the replication of influenza A virus, measles virus, herpes simplex virus types 1 and 2, or human cytomegalovirus. Two other benzodithiin derivatives [1,4-dihydro-6-methyl-2,3-benzodithiin (RD3-0270) and 1,4-dihydro-5-methyl-1-2,3-benzodithiin (RD3-0284)] also inhibit RSV replication at a selectivity index greater a factor of 20. These results suggest that the benzodithiin skeleton is an important structure for inhibitory activity against RSV replication.

We have examined a novel betain-type fluoroalkylated oligomer, RD6-2198, for its inhibitory effects on the replication of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) and respiratory syncytial virus (RSV) in cell cultures. We have found that the compound is a potent and selective inhibitor of these viruses. RD6-2198 inhibited the replication of HIV-1IIIB at a concentration of 0.85 microg/ml with a selectivity index greater than 59 in MT-4 cells. Furthermore, its 50% effective concentration (EC50) values for HSV-1, HSV-2 and RSV, were 0.51, 0.94 and 3.0 microg/ml, respectively. We found that the RD6-2198 suppressed the gp120-CD4 interaction (as monitored by an enzyme-linked immunosorbent assay (ELISA) method). RD6-2198 also inhibited the binding of anti-gp120 monoclonal antibody to gp120 expressed on MOLT-4/IIIB cells (MOLT-4 cells chronically infected with HIV-1IIIB). However, the compound did not inhibit the interaction of anti-CD4 antibody with CD4. These results suggest that RD6-2198 interacts with the viral envelope glycoprotein and thereby inhibits the viral adsorption process. In addition, RD6-2198 was also found to suppress the proliferation of MOLT-4/IIIB cells. When applied topically, RD6-2198 at a concentration of 10 mg/ml completely protected mice from an intravaginal HSV-2 infection.

Both the NS3 protease and the NS4A protein are required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. The NS3 protease domain was fused at its C-terminal end with full length NS4A and expressed in Escherichia coli. This protein (NS3 delta-NS4A) was purified to apparent homogeneity after refolding from extracts recovered from inclusion bodies. During the expression and purification process, NS3 delta-NS4A was not auto-processed in either a cis or trans manner at NS3/NS4A junction site. When the kcat/K(m) values and thermostability of NS3 delta-NS4A were compared with those for maltose binding protein-NS3 fusion protein (MBP-NS3), which contains only NS3 region, the single-chain NS3 delta-NS4A showed enhanced proteolytic activities and thermostability.

We studied the effects of two antiviral agents, human interferon-alpha (IFN-alpha) and ribavirin, on subacute sclerosing panencephalitis (SSPE) virus infections in hamsters. By intracranial administration, IFN-alpha alone improved the survival of infected hamsters by 20% at a dose of 6 x 10(4) IU/kg every other day for 10 days. When the dose of IFN-alpha was increased incrementally to 6 x 10(6) IU/kg, the survival rate increased by 70% in a dose-dependent manner. The combination of IFN-alpha and ribavirin had a synergic inhibitory effect on the replication of SSPE virus in cell culture. Combination of IFN-alpha (at a dose of 6 x 10(5) IU/kg) with ribavirin (at a dose of 1 mg/kg) completely prevented mortality. This was significantly better than either IFN-alpha or ribavirin monotherapy (p < 0.05). Under the conditions used, IFN-alpha did not enhance the toxicity of ribavirin in hamsters. Intraventricular administration of high dose IFN-alpha and ribavirin may have potential usefulness in the treatment of patients with SSPE.

This study evaluated the inhibitory effects of thiazolidine derivatives on hepatitis C virus (HCV) protease and other human serine proteases. The inhibition efficacy was tested with a reversed-phase high-performance liquid chromatography (HPLC) assay system using a NS3-NS4A fusion protein as the HCV protease and a synthetic peptide substrate that mimics the NS5A-5B junction. Nine thiazolidine derivatives showed more than 50% inhibition at 50 microg/ml. The most potent derivative was RD4-6250, with 50% inhibition at a concentration of 2.3 microg/ml; this concentration was lower than those of other protease inhibitors reported previously. The most selective derivative was RD4-6205, with 50% inhibition at a concentration of 6.4 microg/ml, a lower concentration than those on other serine proteases (chymotrypsin, trypsin, plasmin, and elastase). These results suggest that the RD4-6205 skeleton is an important structure for inhibitory activity on the HCV protease NS3-NS4A., (Copyright 1997 Academic Press.)

In the search for effective Tat-dependent transcription inhibitors using a screening assay system that has recently been developed, 2-glycineamide-5-chlorophenyl 2-pyrryl ketone (GCPK) has proved to be a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) replication in vitro. This compound was inhibitory to HIV-1 replication in both acutely and chronically infected cells. The 50% effective concentration (EC50) of GCPK in acutely infected MOLT-4 and CEM cells was 0.62 and 0.13 microgram/ml, respectively. These values were similar to those of the known Tat-dependent transcription inhibitors Ro5-3335 and Ro24-7429. Like these inhibitors, GCPK could inhibit HIV-1 replication in MOLT-4/IIIB (MOLT-4 cells chronically infected with HIV-1) and tumor necrosis factor-alpha-(TNF-alpha)-induced viral activation in OM10.1 cells (a HL-60 clone latently infected with HIV-1). GCPK is distinct from Ro5-3335 and Ro24-7429 in that this novel Tat-dependent transcription inhibitor has no benzodiazepin ring.

The hepatitis C virus (HCV) genome contains the code for a conserved, serine-type protease, called NS3, for the processing of the non-structural protein region of the viral polyproteins. Furthermore, a related protein, NS4A, is an effector or cofactor of NS3 protease activity in the cleavage of NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. To establish an in vitro assay system for the screening of those enzyme inhibitors that inhibit the protease NS3-4A, we prepared a maltose-binding protein-NS3-NS4A fusion protein and a synthetic peptide substrate that mimics the NS5A-5B junction. Cleavage of the synthetic peptide was analyzed by reversed-phase high performance liquid chromatography (HPLC). We showed that the enzymatic activity of the NS3-NS4A fusion protein was enhanced in comparison to the NS3 protein alone. The assay conditions for optimum NS3-4A protease activity were determined to be pH 7.6 and 37 degrees C. In addition, we evaluated several protease inhibitors using the same HPLC assay system. The activity of HCV protease NS3-4A was inhibited by 2714.4 microM diisopropyl fluorophosphate, 270.8 microM N-tosyl-L-lysyl chloromethyl ketone, and 825.5 microM chymostatin. The results of the present study indicated that the synthetic peptide substrate and HPLC assay system are suitable for studying HCV protease activity and may facilitate the development of anti-HCV therapeutic reagents.

We established a sensitive and accurate method for screening of anti-adenovirus agents using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. MKN-28 cells, which are well-differentiated stomach adenocarcinoma cells, were used for adenovirus (ADV) infection and examined for the anti-ADV activities of several established anti-herpes virus agents. ADV-11 is the causative agent of respiratory and urinary infections. It frequently causes hemorrhagic cystitis in immunocompromised hosts. One laboratory strain and 4 clinical isolates of ADV-11 were examined, and found susceptible (in order of decreasing activity) to 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S-2242), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine[(S)-HPMPA ], and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine[(S)-HPMPC++ +]. On the other hand, ganciclovir and iododeoxyuridine were only weakly effective and dextran sulfate was ineffective. Our findings indicate that the MTT assay using MKN-28 cells is applicable to anti-ADV screening. The anti-ADV activity of (S)-HPMPA and (S)-HPMPC was confirmed, and, furthermore, S-2242 emerged as a highly potent and selective inhibitor of ADV-11.

Nucleotide heterodimers were synthesized and examined for their inhibitory effects on the replication of human immunodeficiency virus type 1 (HIV-1), including HIV-1 reverse transcriptase (RT) inhibitor-resistant mutants. 3'-Azido-3'-deoxythymidilyl-(5')-phospho-(5')-6-[(3', 5'-dimethylphenyl)thio]-5-ethyl-1-[(2-hydroxyethoxy)methyl]uracil (AZT-P-E-HEPU-dM) and 3'-azido-3'-deoxythymidilyl-(5')-phospho-(5')-2', 3'-dideoxyinosine (AZT-P-ddI) proved to be highly potent and selective inhibitors of HIV-1 (IIIB strain) in MT-4 cells. The mechanism of inhibition by these heterodimers may be attributed to their degradation and the formation of each constituent. AZT-P-E-HEPU-dM was also markedly inhibitory to an AZT-resistant mutant (HIV-1-IIIB/AZT) and an E-HEPU-dM-resistant mutant (HIV-1-IIIB-R). However, AZT-P-ddI was found to have a less inhibitory effect on HIV-1-IIIB/AZT than on HIV-1-IIIB. The heterodimers of (5',5') AZT and ribavirin (AZT-P-Ribavirin) and (5',5') ddI and ribavirin (ddI-P-Ribavirin) were also synthesized: AZT-P-Ribavirin inhibited HIV-1 replication, but ddI-P-Ribarvirin did not.

The structure-activity relationship of the non-nucleoside HIV-1-specific reverse transcriptase (RT) inhibitors 4-phenyl-1,2,5-thiadiazol-3-yl N,N-dialkylcarbamate (TDA) derivatives was investigated with respect to their anti-HIV-1 activity, RT inhibition, and lipophilicity. 4-Phenyl-1,2,5-thiadiazol-3-yl N,N-dimethylcarbamate inhibited HIV-1-induced cytopathic effect (CPE) by 50% at a concentration of 28.8 microM in MT-4 cells. The activity increased more than 100-fold when the hydrogens at the 2-position and the 6-position in phenyl moiety were substituted by chlorines. However, the derivative with a chlorine at the 4-position of phenyl moiety did not show any inhibition of HIV-1 replication at its non-toxic concentrations. All of the 4-(2,6-dichlorophenyl)-1,2,5-thiadiazol-3-yl N-methyl-N-alkylcarbamates proved inhibitory to HIV-1 replication in the nanomolar concentration range. The TDA derivatives that showed anti-HIV-1 activity also inhibited RT activity in an enzymatic assay. However, the TDA derivatives did not show any specific inhibition of a non-nucleoside RT inhibitor (NNRTI)-resistant mutant and its RT activity. When the TDA derivatives were examined for their inhibitory effect on HIV-1 replication in the presence of 50% human serum, the activity significantly decreased depending on-their lipophilicity.

A marine microalga, Cochlodinium polykrikoides, produces extracellular sulfated polysaccharides. Isolation and purification of the polysaccharides were accomplished by precipitation with ethanol and Cetavlon, followed by DEAE-cellulose column chromatography (polysaccharides A1 and A2). These polysaccharides, which were homogeneous when analysed by both ultracentrifugal and electrophoretic methods, were composed of mannose, galactose, glucose and uronic acid, together with sulfate groups (S = 7-8% w/w). Both A1 and A2 inhibited the cytopathic effect of influenza virus types A and B in MDCK cells, that of respiratory syncytial virus types A and B in HEp-2 cells, that of human immunodeficiency virus type 1 in MT-4 cells; and, except A1 for herpes simplex virus type 1 and A2 for parainfluenza virus type 2 in HMV-2 cells, the cochlodinium polysaccharides showed no antiviral activity against parainfluenza virus types 2 and 3, measles virus, mumps virus or herpes simplex virus type 1 in HMV-2 cells. No cytotoxicity for host cells was observed with these polysaccharides at a concentration of 100 micrograms ml-1. Inhibitory effects on various viruses were achieved at concentrations that were not markedly inhibitory to the blood coagulation process.

We have analyzed mutations in the thymidine kinase (TK) gene of varicella zoster virus (VZV) which showed resistance to 5-iodo-2'-deoxyuridine (IDU) and 5-bromo-2'-deoxyuridine (BrDU). Through sequencing of the TK gene, we found three amino acids were exchanged (41 Asn-->Ser, 266 Cys-->Ile, 288 Ser-->Leu). These mutations were not located at either the nucleoside- or the ATP-binding site. This result suggests that the resistance to IDU and BrDU in this particular strain is due to the change in conformation of TK rather than the replacement of amino acids in the binding sites.

We compared several sialytransferase activities related to synthesis of O-linked and N-linked sialyglycoproteins in Ehrlich ascites tumor cells that grow normally in murine ascites, but are not adherent nor grow in tissue culture (na-EAT cells), with those in cells that were selected to grow in tissue culture and adhere to extracellular matrices (a-EAT cells). Crude Golgi preparations from both cell types contained predominantly beta-D-Gal-(1-->3)-D-GalNAc alpha-(2-->3)-sialyltransferase activity. Sialylation of N-acetyllactosamine, lacto-N-tetraose, and benzyl alpha-D-GalNAc occurred at from 1 to 4% of that activity. Analysis, by ion-exchange HPLC at high pH, of sialylated N-acetyllactosamine showed that na-EAT cells sialylated beta-D-Gal-(1-->4)-D-GlcNAc mostly by alpha-(2-->3)-sialyltransferase, whereas beta-D-Gal-(1-->4)-D-GlcNAc alpha-(2-->6)-sialyltransferase activity was prominent in a-EAT cells. In addition, preparations from na-EAT cells formed significant quantities of an unknown tritiated product from CMP-[9-3H]sialic acid, suggesting at least one other difference in enzyme levels between the cell types. a-EAT cells reestablished in murine ascites for 11 passages retained the sialyltransferase levels characteristic of a-EAT cells. When viable cells were labeled with D-[3H]glucosamine, na-EAT cells formed larger amounts of sialic acid in O-linked glycoproteins than did a-EAT cells.

Several naphthalenedisulfonic acid derivatives were found to be selective inhibitors of human cytomegalovirus (CMV) replication in MRC-5 cells. Among the test compounds, the bis-naphthalenedisulfonic acid derivative having an hexamethylene spacer emerged as the most potent inhibitor of CMV replication. Its 50% antivirally effective concentration (EC50) for AD-169 strain was 12 microM, whereas the compound did not affect the growth of mock-infected MRC-5 cells at concentrations up to 500 microM. The naphthalenedisulfonic acid derivatives were also inhibitory to CMV clinical isolates. Virus yield reduction assay revealed that the compounds significantly reduced virus growth in CMV-infected MRC-5 cells. The bis-naphthalenedisulfonic acid derivatives with an hexamethylene spacer suppressed the expression of CMV-induced immediate early, and early antigens at a concentration of 20 microM, whereas the anti-CMV nucleoside ganciclovir did not do so even at the concentration that was 10-fold higher than its EC50 for CMV-induced plaque formation. Furthermore, naphthalenedisulfonic acid derivatives had to be present at the time of virus infection to exert their anti-CMV activity. These results suggest that the compounds are targeted at an early event in the virus replicative cycle, presumably, virus adsorption.

Complete primary structures of five allergenically active oligosaccharitols (HPG-beta 2-N5a, -N6, -N7a, -N7b, and -N9) derived from a sea squirt H-antigen were studied. Structural characterization was carried out by a new method in which products of limited periodate oxidation, followed by derivatization with p-aminobenzoic acid ethyl ester, were analyzed by a combination of HPLC, fast atom-bombardment mass spectrometry, sequential glycosidase digestion, methylation analysis, and 500-MHz 1H NMR. Established structures of GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3) GlcNAc beta 1-3GalNAc-ol, GalNAc beta 1-4GlcNAc beta 1-3 (GalNAc beta 1-4GlcNAc beta 1-6) GalNAc-ol, GalNAc beta 1-4GlcNAc beta 1-3[GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6] GalNAc-ol, GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-3[GalNAc beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6] GalNAc-ol, and GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3)GlcNAc beta 1-3 [GalNAc beta 1-4 (GalNAc alpha 1-2Fuc alpha 1-3)GlcNAc beta 1-6]GalNAc-ol are represented by HPG-beta 2-N5a, -N6, -N7a, -N7b, and -N9, respectively. These structures have not been encountered previously. Oligosaccharide units GalNAc alpha 1-2Fuc alpha 1-, GalNAc beta 1-4GlcNAc beta 1-, and Fuc alpha 1-3GlcNAc beta 1- are considered to be the allergenically specific epitopes. Partial assignments of 500-MHz 1H NMR spectra of these novel O-linked oligosaccharitols were attempted.

1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) has recently proved to be a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. Combinations of HEPT and recombinant alpha interferon (IFN-alpha) synergistically inhibit the replication of HIV-1 in MT-4 cells at non-toxic concentrations. Synergistic inhibition of HIV-1 replication has also been observed in peripheral blood lymphocytes. These results indicate that the combination of HEPT with IFN-alpha should be further pursued in the treatment of retrovirus infections [i.e. acquired immune deficiency syndrome (AIDS)].

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.

Protein kinase C(PKC) activity in macrophages and polymorphonuclear leukocytes was assayed in beige mouse, the model of Chediak-Higashi syndrome, control C57BL/6 and the heterozygous (+/bg) mice. Regarding enzyme activity in the cytosolic and membrane fractions of these cells, there was no difference between beige mouse and the control. After short-term activation by TPA, the translocated membrane-bound PKC activity in beige mouse decreased rapidly compared with that in control mouse. However, the cytosolic PKC activity decreased at just the same pace as the control. The change in [3H] PDBu binding paralleled the changes in PKC activity. An increase in Ca2+/phospholipid-independent protein kinase by TPA was notable in the membrane fraction of beige mouse. The increase in the kinase activity was abolished and the PKC activity recovered to normal level by the addition of calpain inhibitor, leupeptin, to the incubation of cells along with TPA. Therefore, these findings suggest that a rapid decrease in membrane-bound PKC activity in beige mouse by TPA stimulation is associated with calpain.

We investigated the role of microfilaments in stimulus-induced translocation of protein kinase C (PKC) in polymorphonuclear leukocytes (PMNs) from C57BL/6 mice. Cytochalasin B and dihydrocytochalasin B almost completely inhibited PKC translocation induced by either TPA or Ca2+ ionophore after pretreatment of cells for 30 min. In addition, ML-9, a potent inhibitor of Ca2+/calmodulin-dependent myosin light chain kinase which regulate microfilament contraction, and a calmodulin antagonist W-7, also inhibited PKC translocation. These findings suggest the possibility that microfilaments are involved in the translocation of PKC.

Glycyrrhizin (GL) achieved a dose-dependent inhibition of the replication of human immunodeficiency virus type 1 (HIV-1) in MOLT-4 (clone No. 8) cells within the concentration range of 0.075 to 0.6 mM. Within this concentration range, GL also effected a dose-dependent reduction in the protein kinase C (PKC) activity of MOLT-4 (clone No. 8) cells. A well-known PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), also proved inhibitory to HIV-1 replication in MOLT-4 (clone No. 8) cells. PKC inhibition may thus be considered as one of the mechanisms by which GL inhibits HIV-1 replication. In addition, GL may also owe its anti-HIV-1 activity, at least in part, to an interference with virus-cell binding, since the compound at 1.2 mM partially inhibited the adsorption of radiolabeled HIV-1 particles to MT-4 cells. At this concentration GL also suppressed giant cell formation induced by co-culturing MOLT-4 (clone No. 8) cells with MOLT-4/HTLV-IIIB cells, whereas the PKC inhibitor H-7 failed to do so.

Glycyrrhizin (GL), one of the plant extracts, was investigated for its antiviral action on the human immunodeficiency virus [HIV (HTLV-III/LAV)] in vitro, using cytopathic effect and plaque forming assay system in MT-4 cells (a HTLV-I-carrying cell line). Cloned Molt-4 cells (clone No. 8), which are sensitive to HIV and fuse to giant cells after infection, were also used as a parameter for cytopathic effect of HIV. GL completely inhibited HIV-induced plaque formation in MT-4 cells at a concentration of 0.6 mM, the 50% inhibitory dose being 0.15 mM. GL completely inhibited the cytopathic effect of HIV and the HIV-specific antigen expression in MT-4 cells at a concentration of 0.3 and 0.6 mM, respectively. Furthermore, GL inhibited giant cell formation of HIV-infected Molt-4 clone No. 8 cells. GL had no direct effect on the reverse transcriptase of HIV. Its mechanism of anti-HIV action remains to be elucidated.