Self-complementary AAV-mediated gene therapy restores cone function and prevents cone degeneration in two models of Rpe65 deficiency.

To test whether fast-acting, self-complimentary (sc), adeno-associated virus-mediated RPE65 expression prevents cone degeneration and/or restores cone function, we studied two mouse lines: the Rpe65-deficient rd12 mouse and the Rpe65-deficient, rhodopsin null ('that is, cone function-only') Rpe65(-/-)::Rho(-/-) mouse. scAAV5 expressing RPE65 was injected subretinally into one eye of rd12 and Rpe65(-/-)::Rho(-/-) mice at postnatal day 14 (P14). Contralateral rd12 eyes were injected later, at P35. Rd12 behavioral testing revealed that rod vision loss was prevented with either P14 or P35 treatment, whereas cone vision was only detected after P14 treatment. Consistent with this observation, P35 treatment only restored rod electroretinogram (ERG) signals, a result likely due to reduced cone densities at this time point. For Rpe65(-/-)::Rho(-/-) mice in which there is no confounding rod contribution to the ERG signal, cone cells and cone-mediated ERGs were also maintained with treatment at P14. This work establishes that a self-complimentary AAV5 vector can restore substantial visual function in two genetically distinct models of Rpe65 deficiency within 4 days of treatment. In addition, this therapy prevents cone degeneration but only if administered before extensive cone degeneration, thus supporting continuation of current Leber's congenital amaurosis-2 clinical trials with an added emphasis on cone subtype analysis and early intervention.

Hypoglycemia leads to age-related loss of vision.

The retina is among the most metabolically active tissues in the body, requiring a constant supply of blood glucose to sustain function. We assessed the impact of low blood glucose on the vision of C57BL/6J mice rendered hypoglycemic by a null mutation of the glucagon receptor gene, Gcgr. Metabolic stress from moderate hypoglycemia led to late-onset loss of retinal function in Gcgr(-/-) mice, loss of visual acuity, and eventual death of retinal cells. Retinal function measured by the electroretinogram b-wave threshold declined >100-fold from age 9 to 13 months, whereas decreases in photoreceptor function measured by the ERG a-wave were delayed by 3 months. At 10 months of age Gcgr(-/-) mice began to lose visual acuity and exhibit changes in retinal anatomy, including an increase in cell death that was initially more pronounced in the inner retina. Decreases in retinal function and visual acuity correlated directly with the degree of hypoglycemia. This work demonstrates a metabolic-stress-induced loss of vision in mammals, which has not been described previously. Linkage between low blood glucose and loss of vision in mice may highlight the importance for glycemic control in diabetics and retinal diseases related to metabolic stress as macular degeneration.

Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.

Effects of anesthesia on the blood glucose of C57/BL6J mice were evaluated under conditions commonly used for testing retinal sensitivity with electroretinographic (ERG) recordings. We evaluated the effects of four anesthetics: nembutal (50 mg/kg), pentothal (100 mg/kg), avertin (240 mg/kg), and ketamine/xylazine (100 mg/kg) using saline as control. We measured blood glucose (BG) levels from tail vein blood before and 15 and 60 min following intraperitoneal injections. Fifteen minutes postinjection, all four anesthetics and saline elevated BG with ketamine/xylazine and avertin having substantially greater effects than nembutal, pentothal, and saline. Only the effects of ketamine/xylazine and avertin persisted throughout the test period. Sixty minutes after injecting ketamine/xylazine BG remained elevated at 400 +/- 42 mg/dl, a 167% increase over preinjection levels. Sixty minutes after injecting avertin BG was 288 +/- 10 mg/dl, a 59% increase over preinjection levels. No sustained elevation in BG was detected 60 min following injection of nembutal, pentothal, or saline. Because BG can affect the amplitude of the ERG, caution should be exercised in the use of ketamine/xylazine or avertin. The choice of anesthesia may also be important in diabetes and metabolism research where changes in blood glucose could impact physiological processes.

Regulatory factors on parathyroid hormone-related peptide production by primary culture of lactating rat mammary gland.

Parathyroid hormone-related protein (PTHrP) is a major cause of humoral hypercalcemia of malignancy, but has also been widely found in fetal and adult non-neoplastic tissues. Lactating mammary gland has been shown to produce large amounts of PTHrP, and high levels of PTHrP have been measured in milk. We have examined the influences of several substances on the secretion of two different forms of PTHrP by primary cultures of mammary cells isolated from lactating rats to examine the regulatory mechanisms of PTHrP production by mammary cells. Primary cultures of mammary cells seeded at a density of 10(5) cells per 35 mm culture dish were grown on collagen gels. First, after cells were left 24 hours for attachment and incubated in 2 % FCS containing medium with for 12 hours, PTHrP (1 - 87) secretions were measured in conditioned medium with hormone supplementation for 1, 24 and 48 hours. Progesterone (10(-7) - 10(-5) mol/l) significantly suppressed PTHrP (1 - 87) secretion in a dose-dependent manner (p < 0.01), while 17beta-estradiol had no influence on PTHrP (1 - 87) secretion. Prolactin, a known stimulator of PTHrP expression in vivo, had no effect in this in vitro model. Second, PTHrP (1 - 34) secretion levels from confluent lactating mammary cells for 24 hours were evaluated. The same results were obtained in the case of PTHrP (1 - 87) secretion from non-confluent cells. Furthermore, dexamethasone (10(-6) mol/l) significantly suppressed PTHrP (1 - 34) secretion (p < 0.01). These results suggest that PTHrP production from the lactating mammary gland is suppressed by progesterone as well as dexamethasone. Progesterone dramatically falls after delivery, thus possibly accelerating PTHrP production by lactating mammary glands and resulting in considerable amounts of PTHrP secreted into the milk.

Protease susceptibility of the Caulobacter crescentus flagellar hook-basal body: a possible mechanism of flagellar ejection during cell differentiation.

When motile swarmer cells of Caulobacter crescentus differentiate into sessile stalked cells, the flagellum is ejected. To elucidate the molecular mechanism of the flagellar ejection, flagellar hook-basal body (HBB) complexes from C. crescentus were purified and characterized. The purified HBBs were less stable against acidic pH or protease treatment than HBBs of Salmonella typhimurium, supporting the view that flagellar ejection from C. crescentus is initiated by destruction of the fragile basal structures. In addition, protease treatment of the purified flagella resulted in the specific digestion of the MS ring complex, revealing for the first time the intact structure of the whole rod.

Inhibition of rubella virus growth by Fungizone.

Fungizone added to agar overlay medium inhibited plaque formation in both size and number by rubella virus in rabbit kidney 13 cells. In the presence of 1 microg/ml of Fungizone, the diameter of the plaques was reduced to one half of that in the absence of the drug, and at 5 microg/ml, plaque formation was inhibited by 80%. When the drug was added to the culture medium, the growth of infectious virus was also inhibited with reduction in the synthesis of envelope glycoprotein E1 and capsid protein C in infected cells.

[Preparation of HPLC test solutions for organic impurities in aluminum lakes of food red no. 40 (allura red AC) and food yellow no. 5 (sunset yellow FCF)].

The HPLC determination of organic impurities in Food Red No. 40 aluminum lakes (R-40Als) as directed by Japan's Specifications and Standards for Food Additives, 7th Ed. (JSFA-VII), has problems, such as reproducibility and low recovery. ICP analyses suggested that the problem was caused by the aluminum in the test solution. In the improved method for preparation of the test solution, aluminum was precipitated as a hydroxide gel by boiling with 1% aqueous ammonia. After centrifugation, the supernatant was used for the HPLC analysis of the organic impurities in the lakes. Recoveries of organic impurities were more than 85% from R-40Al spiked at the 0.1 and 1.0% levels of R-40. The proposed method was also adapted for Food Yellow No. 5 aluminum lakes.

Structural determination of the subsidiary colors in food blue No. 1 (brilliant blue FCF) aluminum lake detected by paper chromatography.

One of eight Food Blue No. 1 aluminum lakes (B-1Als) used in the official inspection of coal-tar colors in fiscal year 1999 had a violet sub-spot during paper chromatography and was rejected. To clarify the orgin of the sub-spot, the violet subsidiary color (Sub-V) was isolated from the sample. On the basis of NMR and MS analyses and ion chromatography, the structure of the subsidiary color was elucidated to be 2-[[4-[N-ethyl-N-(3- sulfophenylmethyl)amino]phenyl][4-hydroxyphenyl]methylio]benzenesulfonic acid. The relative content of Sub-V to that of m,m-B-1 in the rejected sample was determined to be 39.5% by HPLC. The relative contents in other submitted samples of B-1Al were in the range of 1.1-3.6%.

[Estimated production by the official inspection of tar colors (including aluminum lakes) in fiscal year 2000].

There were 176 official inspections of tar colors and their lakes in fiscal year 2000, and 175 samples were qualified. The quantity of tar colors that passed inspection in Japan in fiscal year 2000 reached 137.5 tons. Tar color production is estimated by month and by manufacturer. The food tar color produced in the largest quantity was Food Yellow No. 4, accounting for 43.4% during this period.

[Studies on rejected food yellow no. 5 (sunset yellow FCF) aluminum lake].

One out of two sunset yellow FCF aluminum lakes (Y-5Als) did not comply with the specifications in JSFA-VII in the official inspection of tar colors in fiscal year 2000. A sub-spot was detected in the paper chromatography test. This rejected sample was analyzed by HPLC for the subsidiary color, raw materials and intermEdiates in Y-5. The sub-spot was identified as sulfanilic acid azo R salt color, and its content was estimated at 4.5% as the content of Y-5 in Y-5Al being 100.0%.

Influence of jam processing on the radical scavenging activity and phenolic content in berries.

Six selected phenolic aglycons (caffeic and ellagic acids, kaempferol, quercetin, myricetin, and morin) in nine types of berries, and their changes as influenced by jam processing, have been evaluated using optimized HPLC with diode-array detection. The berry samples, fresh and after jam processing, were analyzed, and the total amounts of selected phenolics as aglycons were identified and determined by acid hydrolysis. Their contents in fresh and jam samples did not indicate appreciable changes; therefore, the influence of jam processing on these selected phenolics in berries was suggested to be small, and was mostly present in berries as several conjugated forms that were glycosylated, esterified, etc., in the samples. The total phenolic content of each sample was also determined by the Folin-Ciocalteu method. The three samples of each berry, namely fresh, jam, and acid hydrolysate of the berry, had similar total phenolic contents. On the other hand, the scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured, and acid hydrolysates showed stronger activity than that of the fresh and jam-processed samples for all of the berry types.

Development of GAP-43 mRNA in the macaque cerebral cortex.

To estimate the extent of axonal growth in various areas of the cerebral cortex, we measured the amount of GAP-43 mRNA in the cerebral cortex of developing macaque monkeys. In four areas, i.e., the prefrontal area (FD delta), the temporal association area (TE), the primary somatosensory area (PC), and the primary visual area (OC), the amount of GAP-43 mRNA was measured from the intermediate fetal period [embryonic day 120 (E120)] to the adult stage. In two other areas, i.e., the parietal association area (PG) and the secondary visual area (OB), the amount of GAP-43 mRNA was measured during the postnatal period. The amount of GAP-43 mRNA was highest at E120, decreased roughly exponentially, and approached the asymptote by postnatal day 70 (P70). The amount of GAP-43 mRNA was higher in the association areas (FD delta, TE, and PG) than in the primary sensory areas (PC and OC) during development and at the adult stage. These findings suggest that axonal growth in the cerebral cortex is most exuberant before or during the intermediate fetal period and approximately ends by P70. Furthermore, axonal growth is evidently more intensive in the association areas than in the primary sensory areas during the stage following the intermediate fetal period.

Parameters for plaque formation in the potency assay of Japanese measles vaccines.

Parameters for plaque formation by measles vaccine strains licensed in Japan were studied. For the plaque test, inoculum volume was one of the critical factors for obtaining an appropriate titre of the sample. A linear relationship between the inoculum volume and the apparent reciprocal titre was discovered, enabling the comparison of absolute titres. Another factor affecting the infectivity was the strain-specific temperature sensitivity in the plaque assay. Although all the vaccine strains tested showed the highest titre at 35 degrees C, the pattern of the temperature sensitivity differed from one strain to another. These factors must be taken into consideration in order to obtain an appropriate titre of a vaccine virus.

Detection and comparison of viral antigens in measles and rubella rashes.

Measles and rubella skin lesions were immunocytochemically compared by the avidin-biotin-peroxidase complex method for detecting viral antigens. Cryostat sections of biopsied specimens of the skin were stained with mouse monoclonal antibodies to P protein of measles virus and to E1 protein of rubella virus. The measles virus antigen was concentrated in the corneal layer and the keratinocytes of the epidermis and in the surface part of the dermis in the biopsy secimens taken within 6 days after the onset of rash. On the other hand, the rubella virus antigen was dispersed in all parts of the dermis and the subcutaneous layer but not in the epidermis in the biopsy specimens taken within 2 days after the onset of rash. The differences in the distribution and density of the viral antigen and in the times of its detection suggest distinct patterns of spread of infection with each virus in the skin.

Improved potency assay of rubella vaccine: parameters for plaque formation.

Parameters for plaque formation by rubella vaccine strains licensed in Japan were studied. Formation of clear and large plaques on RK13 cells depends on several essential parameters. Plaques differed in morphology among five vaccine strains and the distinctiveness of the plaques was affected by pH of the agar overlay medium during incubation at 35 degrees C. Plaques became progressively larger in size as the concentration of sodium bicarbonate in the agar overlay medium increased from 0.04% to 0.15%, but the contrast of plaques to the background cells decreased markedly. The most distinct plaques of all vaccine strains were formed in the agar overlay medium containing 0.07% of sodium bicarbonate, i.e., pH 6.83, incubated in a humidified atmosphere containing 5% CO2. The number of plaques formed by vaccine strains decreased at 37 degrees C. Vaccine strains other than MEQ11 and TCRB19 formed larger and more contrasted plaques with sharp outline at 35 degrees C than at 32 degrees C. MEQ11 and TCRB19 strains yielded higher infective virus titres at 32 degrees C, but they formed distinct plaques at 35 degrees C and 32 degrees C. For the plaque test, the inoculum volume was another critical factor for obtaining an approximate titre that reflected the absolute titre of the sample. A volume of 0.1 ml was feasible for a well with a diameter of 35 mm.

Plaque formation of Newcastle disease virus in primary chicken kidney cells.

Using primary chicken kidney (PCK) cells, a plaque assay was carried out with an avirulent strain of Newcastle disease virus (NDV) without adding trypsin to the agar overlay medium. The plaque assay was done in less than 4 days in PCK cells, by 3 days earlier than in primary chicken embryo (CE) cells maintained in the presence of trypsin. The curves of the progeny virus production began to rise 6 h after the infection and reached a plateau at 12 h. Equal amounts of virus were produced in PCK cells both in the presence and absence of trypsin in the culture medium. Viral peptide analysis revealed that a large portion of the HN and F precursor envelope glycoproteins of the NDV-Ulster strain remained uncleaved in PCK-grown virions. This suggests that a marginal proteolytic cleavage of these glycoprotein suffices for the full growth of the progeny virus in PCK cells. A higher shut-off in the host protein synthesis occurred in the virus-infected PCK cells than in the infected CE cells.

Protective effect of monoclonal antibodies to Newcastle disease virus in passive immunization.

A series of monoclonal antibodies (MAbs) against the haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins and the matrix (M) protein of Newcastle disease virus (NDV) were tested for protective effects in passive immunization of newborn chickens against challenge with a virulent heterologous strain of NDV (Italien). MAbs with high virus-neutralizing activity directed to one antigenic site of the HN protein delayed virus growth and significantly prolonged survival time, but all chickens eventually succumbed to infection. MAbs directed to two antigenic sites of the F protein completely suppressed virus growth and prevented death of chickens, although the neutralizing activities of these anti-F MAbs were lower than those of the above anti-HN MAbs. Combined administration of the anti-HN and anti-F MAbs had a synergistic protective effect, but no protective effects were shown by MAbs against the M protein.