Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Method of separating mouse platelets by discontinuous sucrose-gradient centrifugation

A discontinuous sucrose gradient was employed in the separation of mouse blood platelets using a modified Booyse method. The platelets of male CD-1 mice aged 8 to 12 weeks were divided into five distinct populations (A, B, C, D & E). Distribution of light to heavy platelets patterns in 10 normal CD-1 mice was demonstrable at; A (S.G. 1.188), as 14.8 +/- 5.6%; B (S.G. 1.199), 44.0 +/- 4.6%; C (S.G. 1.207), 24.1 +/- 3.4%; D (S.G. 1.214), 13.0 +/- 3.6%; and E (S.G. 1.221), 4.0 +/- 1.5%.     

Biological properties, phage typing and antimicrobial susceptibility of Staphylococcus aureus isolated from dermatitis in laboratory mice

The characteristics of 167 isolates of S. aureus from 106 mice suffering dermatitis were examined. All 167 isolates coagulated both rabbit and human plasmas and 161 of them also coagulated bovine plasma. All the isolates produced heat-stable and heat-labile DNase, phosphatase and yellow pigment, reduced nitrate, hydrolysed egg yolk, Tween 80, and hippurate, and grew on crystal violet agar in colonies of the negative type C and on medium with 10% NaCl. The majority of them produced fibrinolysin, protease and acetoin. Fifty-three percent were gelatinase positive. In hemolysis tests, 25, 57 and 45 isolates showed alpha-, beta-, alpha beta-hemolysis, respectively. Forty isolates did not produce hemolysins in the rabbit and sheep blood agar. All of 75 isolates tested produced acid from fructose, galactose, glucose, glycerol and mannose, but did not from arabinose, dextrin, inulin, raffinose, salicin, sorbitol and xylose. Most of these isolates produced acid from lactose, mannitol, sucrose and trehalose. All of the 75 isolates were highly sensitive to penicillin, methylphenylisoxazolyl penicillin, erythromycin, spiramycin, lincomycin, chloramphenicol, tetracycline, kanamycin, gentamicin and cephaloridine, but were resistant to sulfisoxazole. With phages of human set, all 167 isolates were typable at 100 X RTD. All but one of the typable isolates belonged to mixed lytic groups. These were I + III (35 isolates), I + M (1), I + III + M (124) and I + II + III + M (6), with long phage patterns. When the 167 isolates were biotyped as described by Hájek and Marsálek [7, 8], 5 belonged to biotype A, 1 to biotype B and 60 to biotype C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects

Isono, Shiroh, John E Remmers, Atsuko Tanaka, Yasuhide Sho,Jiro Sato, and Takashi Nishino. Anatomy of pharynx in patients with obstructive sleep apnea and in normal subjects.J. Appl. Physiol. 82(4):1319-1326, 1997.Anatomic abnormalities of the pharynx arethought to play a role in the pathogenesis of obstructive sleep apnea(OSA), but their contribution has never been conclusively proven. Thepresent study tested this anatomic hypothesis by comparing themechanics of the paralyzed pharynx in OSA patients and in normalsubjects. According to evaluation of sleep-disordered breathing (SDB)by nocturnal oximetry, subjects were divided into three groups: normalgroup (n = 17), SDB-1(n = 18), and SDB-2(n = 22). The static pressure-arearelationship of the passive pharynx was quantified under generalanesthesia with complete paralysis. Age and body mass index werematched among the three groups. The site of the primary closure was thevelopharynx in 49 subjects and the oropharynx in only 8 subjects.Distribution of the location of the primary closure did not differamong the groups. Closing pressure(PC) of the velopharynx forSDB-1 and SDB-2 groups (0.90 ± 1.34 and 2.78 ± 2.78 cmH₂O, respectively) wassignificantly higher than that for the normal group (3.77 ± 3.44 cmH₂O;P < 0.01). Maximal velopharyngealarea for the normal group (2.10 ± 0.85 cm²) was significantly greaterthan for SDB-1 and SDB-2 groups (1.15 ± 0.46 and 1.06 ± 0.75 cm², respectively). Theshape of the pressure-area curve for the velopharynx differed betweennormal subjects and patients with SDB, being steeper in slope nearPC in patients with SDB.Multivariate analysis of mechanical parameters and oxygen desaturationindex (ODI) revealed that velopharyngealPC was the only variable highly correlated with ODI. VelopharyngealPC was associated withoropharyngeal PC, suggestingmechanical interdependence of these segments. We conclude that thepassive pharynx is more narrow and collapsible in sleep-apneic patientsthan in matched controls and that velopharyngeal PC is the principal correlate ofthe frequency of nocturnal desaturations.

     

Effect of age on bacterial translocation from the gastrointestinal tract in mice.

To clarify the effects of age on bacterial translocation from the gastrointestinal tract, mice at the age of 1, 2, 4, 6, 12, and 15 months were antibiotic-decontaminated for 4 days and then inoculated orally with streptomycin-resistant Escherichia coli C25. Mice treated with cyclophosphamide and untreated controls were tested for bacterial translocation to the mesenteric lymph nodes (MLN) 2 days later. The population levels of E. coli C25 in cyclophosphamide-treated and untreated mice were approximately 10(9.3) and 10(9.5) per gram of cecum, respectively, at each tested age. There were no significant differences in the incidence of translocation of E. coli C25 to MLN at any of the tested ages, whereas the number of E. coli C25 detected in MLN was higher in young mice than in aged mice in both the cyclophosphamide-treated and untreated groups. These findings suggest that bacterial translocation from the GI tract may be a more important problem in young animals than in aged animals.     

Hematological and clinico-biochemical characteristics of leukemia in Fischer 344 rats.

Seventy-one male and 52 female F 344 rats with leukemia used as controls in the 30-month inhalation studies were characterized by hematological and clinico-biochemical findings. Hematological findings revealed that the leukocyte count, mean corpuscular volume, and mean corpuscular hemoglobin increased in both sexes of leukemic rats showing profound anemia, while the platelet count, erythrocyte count, hematocrit, and hemoglobin concentration decreased. In these rats, the serum levels of low density lipoprotein, free cholesterol, total bilirubin, blood urea nitrogen, and triglyceride and the activities of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, creatine phosphokinase, alkaline phosphatase, and lactate dehydrogenase increased markedly and the level of high density lipoprotein, the oxygen partial pressure, and the cholinesterase activity decreased. Clinical signs such as decrease in redness of the eyes, decrease in body weight, abdominal distension, staining of the public region, and debility were seen in most leukemic animals. These clinical signs and hematological and clinico-biochemical findings may be helpful in diagnosis of leukemia in long-term experiments.     

Biological effects of exposure to high frequency electromagnetics on rabbits and guinea pigs]

To investigate the biological effects of exposure to feeble high frequency electromagnetism, skin surface temperature, blood vessel (arterioles and venules) diameter were examined, using infrared thermography, a laser doppler flowmeter, and a video microscope, respectively, in the ear of rabbits. After exposing the ear of rabbits to high frequency electromagnetism value of 9 MHz for 15 minutes, continued rising of local temperature was demonstrated. Though dilatation of arterioles was not seen. In addition, venules tended to dilate and blood flow also to increase, and microcirculation was accelerated at the site where electromagnetism was exposed. Hazardous effects of long term exposures of high frequency electromagnetism (9 MHz for 30 days, 8 hours/day) on guinea pigs were not observed in their behavior, food consumption, body and organ weights, hematological and biochemical values, macroscopic and microscopic findings on autopsy.     

Prevalence of feline viral antibodies in random-source laboratory cats

Over a period of 1973 to 1979, a serologic survey of virus infections was conducted on feline sera collected in four universities which located in different prefectures; Obihiro, Saitama, Kanagawa and Tokyo. A significant hemagglutination-inhibition (HI) antibody titer of 1 : 8 or higher to feline panleukopenia virus (FPLV) was detected in 130 (58%) of the 226 sera used. No remarkable difference in the HI antibody prevalence in cats to FPLV was recognized by years or localities. Of a total of 188 cats tested, 99 (53%) presented positive serum neutralizing (SN) antibody titers to the No. 1 strain of feline calicivirus (FCV). Especially in Kanagawa, 17 (77%) of the 22 cats had positive SN titers. However, only 42 (22%) of the 188 sera showed positive SN titers to the Kyoritsu strain of FCV. Such lower positivity in the cats was observed with 13% in the SN test to human reovirus type 3 (Reo-3). The incidence of positive SN antibodies to feline rhinotracheitis virus (FRV) also remained in low values of 20 to 27% with the exception of high percentage of 86 in Tokyo. The dissemination of FPLV, FRV, FCV and Reo-3 was briefly discussed in relation with the age distribution of viral antibodies in cats.