Binding characteristics of bovine lactoferrin to the cell surface of Clostridium species and identification of the lactoferrin-binding protein.

The binding characteristics of bovine lactoferrin (bLf) to cells of the Clostridium species were observed by using a horseradish peroxidase-bLf conjugate. A bLf-binding protein (BP) having a relative molecular mass of about 33 kDa was confirmed in the surface layer components from 7 strains of the Clostridium species. The binding of the conjugate to bLf-BP or C. perfringens was strongly blocked by intact Lfs, lysine or arginine residues modified bLf, and deglycosylated bLf, but was not by other milk proteins or by the constituent sugars of glycan. Bacterial growth was inhibited by bLf, but was slightly inhibited by lysine residues modified bLf or deglycosylated bLf. Lactoferricin B did not block the binding of the conjugate, but strongly inhibited the bacterial growth. This suggests that the lysine or arginine residues and glycan of bLf hardly participated in binding bLf to the bacterial cells, but that the amino acid residues and glycan played an important role in inhibiting the growth of bacteria.

Binding of lactoferrin to bacterial cells of the Clostridium species and their agglutination.

Cell agglutination in cell suspensions of 10 strains of Clostridium by lactoferrin (Lf) was observed by obtaining the ratio of increased absorbance (RIA) at 450 nm. The RIA values were very different among the species, being higher in the cell suspensions with bovine Lf (bLf) than in those with human Lf. The binding ability of bLf to the bacterial cells was also observed by an enzyme-linked ligand-binding assay, using the conjugate of iron-free or iron-saturated bLf with horseradish peroxidase (HRPO). The binding ability of bLf was very different among the 10 species, and showed a significant correlation with the cell agglutination of each strain. bLf formed a complex with the cells of C. perfringens, did not dissociate in 2 M NaCl or 4 M urea, but did dissociate in 1 M KSCN. These results suggest that the agglutination of cells of the Clostridium species by bLf is probably caused by the cooperative action of at least electrostatic and hydrophobic interactions between bLf and certain components of the cell surface.

[Age-related changes in the retina of WBN/Kob rats--a pathological study].

Male rats of the WBN/Kob strain, which are known to spontaneously develop diabetes with aging, were examined for histopathological changes in the retina. Five rats (10 eyes) each of WBN/Kob and Wistar/ST as a control were used, and the thickness of the retinal layers, both the central region and the peripheral region of the retina, were measured on weeks 1, 2, 3, 5, 9, 13, 17, 23, 27, 36, 45, 54, 67 and 80 after birth. The rod and cone cell layer in WBN/Kob rats was under-grown, and its thickness decreased 71.7% in the central zone and 59.3% in the peripheral zone of the retina compared with that of the control. In the central and peripheral retina, the rod and cone cell layer, outer nuclear layer and outer plexiform layer, as well as the inner plexiform layer in the central retina, gradually decreased in thickness from 5-45 weeks of age. In the central and peripheral parts of the retina, the number of nuclei decreased in accordance with the thinning of the nuclear layer. The thinned layers showed only cell loss. The rod and cone cell layer of the peripheral retina was thinner than that of the central retina. We obtained the following findings in the retina of male WBN/Kob rats. First, the rod and cone cell layer is undergrown compared with that of the control. Second, the first change occurred in the rod and cone cell layer. Third, the thinning of the rod and cone cell layer appeared at 5 weeks of age, and thinning with aging was slow. And finally, the thinning of the peripheral retina was more severe than that of the central retina. From the above findings, it seems that retinal changes in WBN/Kob rats are similar to the retinal degeneration of rds mice (retinal degeneration slow mice) and that WBN/Kob rats provide a useful animal model for human retinopathy.

Ceruloplasmin concentration in human colostrum and mature milk.

The concentration of ceruloplasmin (CP) in human milk was obtained by measuring the increased absorbance of a solution containing apotransferrin and Fe2+ at 460 nm after adding the milk. The CP concentrations in colostrum and mature milk at less than 1 month after parturition were 4.45 +/- 1.23 mg and 4.09 mg +/- 1.47 mg/100 ml, respectively. In mature milk after more than one month, the CP concentration had decreased to 1.72 +/- 0.91 mg/100 ml.

Interaction of lactoferrin with ascorbate and the relationship with bleomycin-dependent DNA damage.

The interaction between bovine lactoferrin (bLf) and ascorbate (Asc) was investigated through malondialdehyde (MAD) formation in a solution containing DNA, bleomycin (BLM), and Fe2+ or Asc. The inhibition by bLf on MDA formation in the presence of Asc was not changed even by adding carbonate or oxalate ions to the solution. The percentage inhibition by the hydrolysates of bLf treated with pepsin, trypsin, and both enzymes on MDA formation was almost the same as that by the untreated bLf in the presence of Asc. The inhibition of MDA formation also occurred with the filtrate obtained from a solution containing bLf and Asc, but not with that from a solution of bovine serum albumin and Asc. The interaction of bLf and Asc was observed by gel filtration in a Sephadex G75 column. The binding amount of Asc was estimated to be 87 mol per mole of bLf.

[Age-related changes in the visual function of WBN/Kob rats--an electrophysiological study].

Changes in the visual function of the male WBN/Kob rat known as diabetic strain with increasing age were studied electrophysiologically. The measurements of electroretinogram (ERG) and visual evoked potential (VEP) in male WBN/Kob rats were performed at 5, 9, 13, 17, 23, 27, 36, 45, 54, 67 and 80 weeks of age, and compared with those in male Wistar/ST rats used as control. The prolonged latencies and decreased amplitudes of a- and b- waves and oscillatory potentials of ERG in WBN/Kob rats aged over 17 or 27 weeks were recorded under the light or after 20 min dark adaptation. The visual abnormality as indicated by non-recordable ERG was obvious in WBN/Kob rats aged 67 and 80 weeks. No changes of N1, P and N2 peak latencies of VEP were recorded in WBN/Kob rats with increasing age. The amplitudes between N1 peaks and P peaks decreased remarkably in WBN/Kob rats aged 54 weeks. The amplitudes of VEP in 67 or 80 weeks old rats in which ERG disappeared showed 26 or 28% of those in 5 weeks old rats. From these results, recording of ERG and VEP suggested that the visual abnormality occurred in male WBN/Kob rats aged 67 or 80 weeks and the process of visual abnormality with increasing age was clarified.

Age-related changes in the visual organ of WBN/Kob rats--an ophthalmoscopical study.

Changes in the visual organ with aging were investigated non-surgically in male WBN/Kob rats, in which spontaneous diabetic mellitus commonly occurs. Male Wistar/ST rats at the ages of 5, 9, 13, 17, 23, 27, 36, 45, 54, 67 and 80 weeks were examined for changes in the peri-orbital region, eyelid, lens, corpus vitreum and ocular fundus by macroscopic inspection and fundoscopy. The area around the eyelid was stained with reddish-brown excretions from the Harderian gland. This stain was frequently observed in rats of both strains aged 54 weeks or over, showing no difference in frequency between WBN/Kob rats and Wistar/ST rats. Lens opacity was observed in male WBN/Kob rats aged 67 weeks or over, and narrowing of the retinal artery and vein in those aged 36 weeks or over. The occurrence of lens opacity was consistent with that of visual dysfunction as determined by a test of the ability to differentiate light from dark using a Y-shaped apparatus (Kiyosawa et al., 1993).

Suppressive Effects of Lactoferrin on Bleomycin-dependent DNA Damage by the Iron Ion and Ascorbate.

The effects of the iron ion and reductones on bleomycin (BLM)-induced DNA degradation were observed by measuring the amount of malondialdehyde (MDA) formed. The formation of MDA was detected after adding Fe(2+) or Fe(3+) + Asc combined to a solution consisting of DNA and BLM, but not after adding Fe(3+) alone, catecholamines or phenolic compounds. Marked suppressive effects of lactoferrin (Lf) and ovotransferrin on MDA formation were observed in the solution with Fe(2+) or Asc. However, serum albumin, ß-lactoglobulin, α-lactalbumin, and ovoalbumin had no effect on suppressing MDA formation. The suppression by bovine Lf (bLf) of MDA formation decreased with increasing iron saturation at 1.0 mm Fe(2+), but at 100% iron saturation, 51.2% of apo-bLf remained suppression. In the solution with 0.25 mm Asc, 100% iron saturated bLf markedly enhanced MDA formation.

[Age-related changes in the visual function of WBN/Kob rats: a behavioral study].

Changes in visual function with increasing age were studied in male WBN/Kob rats, a new diabetic strain. In order to detect visual abnormalities in WBN/Kob rats, 5 WBN/Kob and 5 Wistar/ST (control) rats from each group were examined at 5, 9, 13, 17, 23, 27, 36, 45, 54, 67 and 80 weeks of age using dark and light distinction and open-field tests. The rates at which WBN/Kob rats selected the dark area were 85.0 to 100%, 58.8% and 52.6% at 5 to 54, 67 and 80 weeks of age, respectively, while those for Wistar/ST rats were 85.0 to 100% in each age group. Visual abnormality was obvious in WBN/Kob rats aged 67 and 80 weeks, compared with Wistar/ST rats (P < 0.05). There were no significant signs of visual abnormality in the open-field test. Detection of visual abnormality seems to be possible from 67 weeks of age.

[Possibility of detecting visual functional disturbance in rats by the open-field test].

In order to determine whether visual disturbance in small rodents is detectable by means of the open-field test, the ambulation scores of enucleated and intact rats in an open-field were measured. In a 30-min test of Slc:Wistar/ST rats, there were significant differences in ambulation scores between enucleated and intact rats in the first 10-min. In addition, the effects of observation times (09:00-11:00, 13:00-15:00 and 17:00-19:00 hr) and strains on the open-field test were studied using a 10-min test. Regardless of observation times, ambulation scores of enucleated rats of the Slc:Wistar/ST and F344/N Slc strains were significantly elevated as compared with those of intact rats from both strains. When tested during the 13:00-15:00 hr period, there were significant differences in the Slc:SD rats. These findings indicate that the changes in ambulation are due to visual disturbance. However, significant ambulatory changes in F344/N Slc rats were documented at the three different observation times. Based on these results, Slc:Wistar/ST rats appear to be suitable for detecting visual disturbance by the open-field test.

[Visual evoked potential from dura in rats].

Visual evoked potentials (VEPs) were recorded from 8 different points (from points A and B near the bregma to points G and H near the occipital bone) of the dura under parietal bone in rats. The study was performed with 68 adult male Slc: Wistar/ST rats weighing 259.9-298.0g. VEPs recorded from the dura had a large negative component with a peak around 45 msec (N1), a large positive component with a peak around 75 msec and a large negative component with a peak around 120 msec (N2). Points A and B were not suitable for recording of the VEPs because some rats showed no N2 peak. The amplitude of peaks N1 to P at point E, upper the left optic layer of the superior colliculus, was higher than those from other points. Point E was the best position for recording of the VEP from dura in Slc: Wistar/ST rats.

[Comparison of the gold wire electrode with cotton wick electrode for electroretinography in small laboratory animals].

Using the gold wire and cotton wick electrodes, the electroretinograms of mice and rats were measured, and then they were compared. The results obtained were as follows: 1) There were no remarkable differences in the latencies and amplitudes of a- and b-waves, amplitudes and numbers of waves of oscillatories between two different electrodes. 2) It was easy to make and maintain the gold wire electrode as compared with the cotton wick electrode. From these results, it appears that the gold wire electrode is useful for measuring the electroretinogram of small laboratory animals.

[The application of industrial endoscope to observation of the ocular-fundus in small laboratory animals].

Observation and recording methods of the ocular-fundus in small laboratory animals were studied using the industrial endoscope and VTR systems, respectively. The ocular-fundus was observed widely, brightly and clearly in the usual animal facility. In addition, the ocular-fundus was recorded easily and it was possible to examine the ocular-fundus recorded by the VTR systems.

Copulatory behavior of sexually inexperienced male voles paired with proestrous females.

The copulatory behavior in sexually inexperienced male voles aged 9-13 weeks old was observed under dim red illumination. Proestrous females were used as stimulus voles in copulatory behavior tests. A 30-min test session was recorded. All males showed at least one ejaculation within 30 min. In addition, the pattern of copulatory behavior in male voles was compared to that reported previously in male hamsters. The frequencies of mount, intromission and ejaculation in male voles were similar to that in male hamsters but there were significant differences in the latencies of mounting, intromission, ejaculation and post-ejaculation between the two species.

p-Chloroamphetamine-induced ejaculation and the 22-kHz vocalization of the male rat.

It is well known that the male rats paired with receptive females emit the 22-kHz vocalization after ejaculation. The question addressed is whether after ejaculation injected with PCA without receptive females the 22-kHz call would be emitted. Emission of the 22-kHz call in males injected by PCA was not observed after ejaculation.

Oxygen uptake activity and aerobic metabolism of Streptococcus thermophilus STH450.

Streptococcus thermophilus STH450 had a very high oxygen uptake. This strain was then compared with aerobic metabolism to S. thermophilus ATCC 19258, a reference strain for aerobic metabolism. Molecular oxygen, which was absorbed by S. thermophilus STH450 during aerobic glycolytic metabolism, was involved in the oxidation of NADH by the catalytic activity of NADH oxidase. The portion of pyruvate that corresponded to the oxidized NADH was committed to form alpha-acetolactate, acetoin, and diacetyl. Both strains were deficient in peroxidase and pyruvate oxidase activities; therefore, NADH oxidase was probably the terminal oxidase in aerobic glycolytic metabolism. Oxygen uptake and NADH oxidase activities were significantly higher in S. thermophilus STH450 than in S. thermophilus ATCC 19258. alpha-Acetolactate, acetoin, and diacetyl also accumulated during aerobic glycolytic metabolism of S. thermophilus STH450. However, when both strains were grown in the presence of pyruvate, these metabolites were equivalent. Hence, less oxygen might be needed for pyruvate metabolism.

Antigenicity of casein enzymatic hydrolysate.

An enzymatic hydrolysate of bovine casein were studied for molecular size and antigenic properties. As determined by Sephadex G-25 gel filtration, polypeptide fractions having molecular weights greater than 1,000 were not detected in the soluble fraction of the casein hydrolysate. Antigenicity of the fraction was not detected by heterologous passive cutaneous anaphylaxis in guinea pig-rabbit antiserum system mediated by immunoglobulin G types of antibody (guinea pig passive cutaneous anaphylaxis) and its inhibition test. Furthermore, it was not detected by rat long latency homologous mediated by immunoglobulin E antibody and its inhibition test. To prepare anticasein immunoglobulin E serum for rat long latency homologous passive cutaneous anaphylaxis, immunization of rats was by single subcutaneous injection of casein with aluminum hydroxide. Antiserum with highest passive cutaneous anaphylaxis titer (1:64) against bovine casein was obtained by a dose of 2,000 micrograms of the protein at day 12.

The content of free and bound inositol in human and cow's milk.

The gaschromatographic method was used for the quantitative analyses of inositol in milk. The content of inositol in human and cow's milk at the different lactation periods was determined. The content of total myoinositol in human milk was 32.7 plus or minus 15.2 mg/100 ml in colostrum, 17.8 plus or minus 1.9 mg/100 ml in transitional milk, and 14.9 plus or minus 3.1 mg/100 ml in mature milk. In cow's milk, it was 10.6 plus or minus 1.0, 7.0 plus or minus 1.1, and 4.1 plus or minus 1.0 mg/100 ml, respectively. These values were very similar to those obtained by the microbiological method. The presence of lipid-bound myoinositol in both kinds of milk was confirmed and the content was 0.22 plus or minus 0.09 mg/100 ml in human milk and 0.36 plus or minus 0.10 mg/100 ml in cow's milk. A small amount of scylloinositol was found in both human and cow's milk, while dextroinositol was not found in either.