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OBJECTIVE: This paper describes the development of a reliable scale of standards for use in evaluating the progress of the transition from milk to solid food in infants and preschool children. The maturation of chewing and swallowing behavior in infants and young children, which enables processing of solid food, varies, and a scale would assist not only in the instruction of mothers and nurses but also in preventing delay in the introduction of solid food. DESIGN: A range of 159 reference foods were selected on the basis of intake during the period of transition from liquid to solid food. These foods were listed in our previous study, Validity and reliability were tested to create a scale. METHODOLOGY: Foods were selected on the basis of 50% of the subjects studied being able to eat them, and on the food groups classified by cluster analyses using the Varclus procedure of SAS. Validity, of the scale was tested by using Pearson's correlation coefficient between the scale score of selected food items and the total score of all 159 food items. The total score of 159 food items was calculated using the general linear models (GLM) procedure of SAS. Reliability was tested using Cronbach's coefficient alpha. SETTING: Public health centers in Aomori, Tokyo, Saitama, Nagano, and Okinawa (Japan). SUBJECT: Five hundred and eighty healthy mothers and children from 2 to 46 months were randomly selected and 470 (81.0%) completed the study. To avoid regional bias, subjects were drawn from northern to southern prefectures in Japan, namely Aomori, Tokyo, Saitama, Nagano, and Okinawa. RESULTS: Twenty food items were selected. By analyzing the score correlation using Pearson's correlation coefficient (R =0.97, P <0.001) and GLM (R2 =0.95, P <0.001), it was confirmed that these 20 food items adequately represented the original 159. The reliability was also found to be sufficient (Cronbach's coefficient alpha=0.96). CONCLUSIONS: The findings demonstrate that a scale of standards for measuring progress in chewing ability can be created using 20 food items. Such a standard will provide a useful basis against which to assess delay of solid food introduction in childhood. SPONSORSHIP: Grant-in-Aid for Scientific Research, provided by the Japanese Ministry of Education, Science and Culture, Project No. 07838030.

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Changes in semen quality of healthy men is a controversial issue throughout the world. It is suspected that many chemical endocrine disrupters may affect the quality of semen. Although exposure to them may be extensive in Japan, no evidence of changes in semen quality has been reported. In this study, changes in semen volume and sperm counts were analyzed over 20 years in the Sapporo area of Japan. Semen volume and sperm counts were measured in 254 and 457 normal, healthy volunteers who lived in the Sapporo area in 1975-1980 and 1998, respectively. Posters and handbills were used to recruit participants in both studies. Semen samples were collected by masturbation after 3 days or more of abstinence. There was no change in semen volume between 1975-1980 and 1998. Mean sperm counts were 70.9 +/- 47.3 x 10(6)/mL in 1975-1980 and 79.6 +/- 49.3 x 10(6)/mL in 1998. Sperm counts did not decline over about 20 years. No significant correlation between age and sperm counts was recognized in either study. The rates of subjects with oligozoospermia and azoospermia were the same in both studies. In the 1975-1980 study, 34 of 254 (13.4%) participants had a child, and in the 1998 study, 51 of 457 (11.2%) participants had a child. Mean sperm count was significantly (P < .02) lower in the earlier study (66.0 +/- 44.9 x 106/mL) than in the 1998 study (98.7 +/- 60.2 x 10(6)/mL). This is the first reliable report in which changes in sperm counts in Japan were studied. We conclude that there was no evidence of deterioration in sperm counts of normal healthy men who lived in the Sapporo area of Japan over 20 years. However, selection bias in the recruitment of volunteers and the issue of variable abstinence might have affected the results of these studies. Therefore, well-designed prospective studies should be performed in several different regions to extrapolate our results on sperm counts to healthy, young Japanese men in general. Key words: Fertility, endocrine disruptors, seminalysis.

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Intestinal Bifidobacterium species are thought to be beneficial in animal and human intestines. We studied the mechanisms of Bifidobacteria in antitumor activity using a cell wall preparation (WPG) of B. infantis (Cancer Res., 45, 1300, (1985)). WPG enhanced the in vitro antitumor activities of mouse peritoneal exudate cells elicited with proteose-peptone (P-PEC) and thioglycollate broth (TG-PEC), determined by cytostatic ([3H]thymidine uptake inhibition) and cytolytic ([3H]uridine release) assays. Tumor necrosis factor-alpha (TNF-alpha) and reactive nitrogen intermediates (RNI) play a role in such augmented cytotoxicity, because anti-TNF-alpha antibody almost completely blocked the increased cytolytic activity of P-PEC in the presence of WPG. Moreover, WPG induced RNI in the supernatant of TG-PEC in a dose-dependent manner. The mRNA expression of several cytokines (IL-1 beta, IL-6, IL-10, IFN-alpha and TNF-alpha) was induced in BALB/c mouse peritoneal cells 3 h after an intraperitoneal injection of WPG (3 h WPG-PEC). However, this expression disappeared from 24 h WPG-PEC, except for that of IFN-alpha. IFN-gamma was not induced. Kinetic studies of the tumor neutralizing activities of the WPG-PECs by means of the in vivo Winn assay revealed that the activity emerged at 1.5 h, became maximal at 3 h and disappeared at 24h. These results indicated that Bifidobacterial WPG is a Biological Response Modifier (BRM) with characteristics similar to those of other bacterial BRMs.

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1. Mutants devoid of lambda- and kappa-toxin and hemagglutinin (HA), respectively, were isolated from Cl. perfringens PB6K. The lambda- and HA- mutants could be classified into a and b groups by complementation but the kappa- mutants were all of the a group. 2. All b group mutants isolated, irrespective of the marker used for isolation, were pleiotropically negative or leaky with respect to theta-, lambda- and kappa-toxin and HA production. 3. Lambda-toxin produced by complementation was proved to be a rennet-like protease. 4. The activities of 12 extracellular enzymes, including sialidase, of several b group strains and the parent PB6K were compared, but no definite differences were observed. From this finding, the productions of these enzymes were concluded not to be regulated by the same mechanism as theta-, lambda- and kappa-toxin and HA. 5. Cl. perfringens CN3870 was also studied. Findings were similar to those on PB6K except for very low activity of HA.

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1. Mutants of a group were converted to b group by a second NG treatment. The resulting b group mutants could not produce the marker products that had been lost on the first NG treatment but could produce the others by complementation. 2. Mutants of b group were converted to constitutive mutants by a second NG treatment. No back mutation from b group was observed.

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The equilibrium and kinetics of thermal unfolding of yeast 5S ribosomal RNA have been studied by optical methods, in a low ionic strength environment without Mg2+, to follow the disruption of the secondary structure base pairs in the molecule. The equilibrium results demonstrated that all of the helical regions melted simultaneously, and the kinetics of the thermal unfolding were first order. These findings suggest the validity of the two-state approximation for the unfolding reaction under the present conditions. The total number of secondary structure base pairs estimated from our experiment was consistent with that contained in the conformational model based on the Raman spectrum rather than that in the one derived by the enzymic digestion method. Taking our results on the kinetic behavior of the thermal unfolding overall, we propose that the 5S RNA has a partly melted secondary structure under the solvent conditions used.

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Five proteinase inhibitors which all inhibit the activity of bovine trypsin [EC 3.4.21.4] were isolated from African Elapid venoms of Hemachatus haemachatus (HHV, Ringhal's cobra) and Naja nivea (NNV, Cape cobra). All the inhibitors were essentially homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecylsulfate. Amino acid analysis and terminal analysis also supported their chemical homogeneities, except for one of the two inhibitors from Hemachatus haemachatus venom. The isolated inhibitors had a molecular weight of about 6,500, consisting of 52 to 57 amino acid residues, and they were all devoid of tryptophan. However, their amino acid compositions differed from each other. One of the three inhibitors isolated from Naja nivea venom, designated NNV inhibitor Ia, was unique, in that 4 half-cystinyl residues per mole fof the polypeptide were present, whereas all the others contained six residues. Of the isolated proteinase inhibitors, the complete amino acid sequences of two major inhibitors were established by manual and automatic Edman degradations and standard enzymatic techniques. Each of the inhibitors, designated HHV inhibitor II and NNV inhibitor II, consisted of 57 amino acid with arginine and glycine at the NH2- and COOH-termini, respectively. Both contained six half-cystines in disulfide linkages, and their overall amino acid sequences were similar, showing 91% homology. The two inhibitors differed in sequence by only five amino acid replacements, Asp-3 to Arg; Tyr-17 to Arg; Leu-25 to Arg; Gln-32 to Glu; and Arg-52 to His, in the 57 residue peptide chain. Comparing the amino acid sequences of these two cobra venom inhibitors with those of Russell's viper venom inhibitor II and bovine pancreatic trypsin inhibitor (BPTI), about 50% homology was found in their sequences. The 6 half-cystinyl residues of these inhibitors were in the same linear positions. Moreover, the regions which are structurally and functionally important in the well-known BPTI molecule were found with extremely high sequence homology in the cobra venom inhibitors. These findings strongly suggest that the cobra venom inhibitors as well as Russell's viper inhibitor II have very similar conformations to that established for BPTI.

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A simple method, involving NAD+-Sepharose chromatography, was developed for the preparation of snake venom phosphodiesterase (EC 3.1.4.1) almost free from 5'-Nucleotidase (EC 3.1.3.5). Using an NAD+-Sepharose 4B column, phosphodiesterase was eluted in the unadsorbed fraction, whereas 5'nucleotidase was strongly adsorbed. The latter enzyme was desorbed when 0.2 M sodium bicarbonate buffer containing 1mM beta-NADH was used as a solvent. The affinity column could be used at least four times without any decrease of potency, and the method was applicable for the preparation of phosphodiesterase from the venoms of rattlesnake (Crotalus adamanteus) and Japanese mamushi (Agkistrodan halys blomhoffi).

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