Reliability, validity, and norms of the 2-min walk test in children with and without neuromuscular disorders aged 6-12.

PURPOSE: The 2-min walk test may be more appropriate functional exercise test for young children. This study aimed to examine the 2-min walk test's reliability; validity; and minimal clinically important difference; and to establish norms for children aged 6-12. METHODS: Sixty-one healthy children were recruited to examine the 2-min walk test's reliability. Forty-six children with neuromuscular disorders (63% cerebral palsy) were recruited to test the validity. The normative study involved 716 healthy children without neuromuscular disorders (male = 51%, female = 49%). They walked at a self-selected speed for 2 min along a smooth, flat path 15 m in length. RESULTS: The mean distance covered in the 2-min walk test was 152.8 m (SD =27.5). No significant difference was found in the children's test-retest results (p > 0.05). The intra- and inter-rater reliability were high (all intra-class correlation coefficients >0.8). All children, except one with neuromuscular disorders, completed the 2-min walk test, of which the minimal clinically important difference at 95% confidence interval was 23.2 m for the entire group, 15.7 m for children walking with aids, and 16.6 m for those walking independently. CONCLUSIONS: The 2-min walk test is a feasible, reliable, and valid exercise test for children with and without neuromuscular disorders aged 6-12. The first normative references and minimal clinically important difference for children with neuromuscular disorders were established for children of this age group. Implications for rehabilitation The 2-min walk test is a feasible, safe, reliable, and valid time-based walk test for children aged 6-12 years. Normative references have been established for healthy children aged 6-12 years. Minimal clinically important difference at 95% confidence interval were calculated for children with neuromuscular disorders who walked without aids (i.e., independent and stand-by supervision) and those who walked with aids equal to 16.6 and 15.7 m, respectively. Distance covered by the healthy children in the 2 min did not correlate with age, gender, height, and weight of the children.

Dynamics of air temperature, velocity and ammonia emissions in enclosed and conventional pig housing systems.

This study aimed to compare the dynamics of air temperature and velocity under two different ventilation and housing systems during summer and winter in Korea. The NH3 concentration of both housing systems was also investigated in relation to the pig's growth. The ventilation systems used were; negative pressure type for the enclosed pig house (EPH) and natural airflow for the conventional pig house (CPH). Against a highly fluctuating outdoor temperature, the EPH was able to maintain a stable temperature at 24.8 to 29.1°C during summer and 17.9 to 23.1°C during winter whilst the CPH had a wider temperature variance during summer at 24.7 to 32.3°C. However, the temperature fluctuation of the CPH during winter was almost the same with that of EPH at 14.5 to 18.2°C. The NH3 levels in the CPH ranged from 9.31 to 16.9 mg/L during summer and 5.1 to 19.7 mg/L during winter whilst that of the EPH pig house was 7.9 to 16.1 mg/L and 3.7 to 9.6 mg/L during summer and winter, respectively. These values were less than the critical ammonia level for pigs with the EPH maintaining a lower level than the CPH in both winter and summer. The air velocity at pig nose level in the EPH during summer was 0.23 m/s, enough to provide comfort because of the unique design of the inlet feature. However, no air movement was observed in almost all the lower portions of the CPH during winter because of the absence of an inlet feature. There was a significant improvement in weight gain and feed intake of pigs reared in the EPH compared to the CPH (p<0.05). These findings proved that despite the difference in the housing systems, a stable indoor temperature was necessary to minimize the impact of an avoidable and highly fluctuating outdoor temperature. The EPH consistently maintained an effective indoor airspeed irrespective of season; however the CPH had defective and stagnant air at pig nose level during winter. Characteristics of airflow direction and pattern were consistent relative to housing system during both summer and winter but not of airspeed. The ideal air velocity measurement favored the EPH and therefore can be appropriate for the Korean environment. Further emphasis on its cost effectiveness will be the subject of future investigations.

Effect of microclimate on particulate matter, airborne bacteria, and odorous compounds in swine nursery houses.

Nursery pigs are vulnerable to environmental risks associated with the microclimate and aerial contaminants. This study was carried out to assess the effect of microclimate (i.e., temperature, relative humidity, and air speed) on the quantity of particulate matter (PM), airborne bacteria, and odorants in nursery houses. Data were collected from 15 farms in different locations throughout South Korea during 4 seasons; daily sampling times were from 1000 to 1100 h in the morning. A nonparametric correlation analysis revealed correlations between microclimate variables and airborne contaminants in different seasons. Over the entire year, negative correlations were observed between temperature, air speed, and some odorous compounds (P < 0.05). Furthermore, negative correlations were observed between temperature, air speed, and relatively large airborne particulates, such as PM(10) (PM mean aerodynamic diameter ≤10 µm), PM(7) (PM mean aerodynamic diameter ≤7 µm), and total suspended particles (P < 0.05). A possible reason for these negative correlations is that increased ventilation at an increased room temperature could transfer most airborne particulates that are carried with odorous compounds out of the nursery houses. On the other hand, because of the sensitivity of coliform bacteria to temperature, positive correlations were observed between temperature and total coliform and Escherichia coli counts (P < 0.01). Because it is a challenging task to control the quantity of aerial contaminants in nursery houses, the relationships between the microclimate and airborne contaminants established in this study could be used to reduce those contaminants by controlling microclimate variables. The correlations established in the current study could also be helpful in establishing guidelines for good management practices in nursery houses.

Prediction of the nutrients value and biochemical characteristics of swine slurry by measurement of EC--electrical conductivity.

Slurry samples, collected from 41 commercial swine farms in South Korea, were characterized in various physico-biochemical, macro and micronutrients, heavy metals and microbial parameters. Interestingly, significant variations were observed in all the parameters. However, positive relationships were noticed between EC and ammonia nitrogen (NH(3)-N), total nitrogen (TN), total potassium (TK), specific gravity (SG), total solids (TS), volatile solids (VS), fixed solids (FS), total dissolved solids (TDS) at R(2)=0.91, 0.74, 0.69, 0.60, 0.50, 0.48, 0.55, and 0.52, respectively. Whereas phosphorous and other nutrients shown poor correlation. Escherichia coli and Salmonella were counted at an average of 5.04 log(10) colony forming unit (CFU)/mL and 3.55 log(10) most probable number (MPN)/mL, respectively. Equations for predicting nutrients content in swine slurries are presented with EC, because it is an easily determinable parameter. The data obtained in this study could be used as a guideline for Good Management Practices in South Korean swine farms as well as other countries.

Molecular cloning and functional study of rat estrogen receptor-related receptor gamma in rat prostatic cells.

BACKGROUND: Based on high homology of ERRs with ERs, we hypothesize that ERRs might functionally cross talk with ERs or independently in prostatic cells. METHODS: We examined the ERRgamma expressions in rat prostates and Nb rat prostate cancer model, and its growth regulation in stable transfectants of prostatic cells. RESULTS: We cloned the ERRgamma cDNA from rat prostate by RACE-PCR. Its expression was confirmed by Northern and immunoblottings. Real-time RT-PCR showed that its expression in castrated prostates was androgen-dependent. ERRgamma was expressed in prostatic epithelial cells, but showed reduced expressions in neoplastic prostates. Transfections confirmed that ERRgamma was expressed in prostatic cells as nuclear protein and transcriptionally active without estradiol. Its overexpression in ERRgamma-stable transfectants of NbE-1 and MAT-Lu cells inhibited their in vitro proliferation, anchorage-independent growth in soft-agar and tumorigenicity in nude mice. CONCLUSIONS: Our studies show that ERRgamma is functionally expressed in rat prostate and may play anti-proliferative actions in prostatic cells. Its co-expression with ERs suggests that besides ERs, ligand-independent ERRgamma is also involved in prostatic growth and functions.

Current status of water reuse systems in Korea.

In Korea, the current water resources will fall short by 2.6 billion tons to meet the 38 billion ton water demand in the year 2020. To overcome the future water shortage, it is desirable to minimize water consumption and to reuse treated wastewater. There are a total of 99 on-site water-recycling systems in the country. The potential capacity of the 99 systems is 429 thousands tons/day, which is 3.6% of the total service water. Compared to other industrialized countries, the number of the water recycling systems in Korea is extremely small. This is mainly due to the following reasons. First, in Korea, any building with more than 60,000 m2 of total floor space is required to install a water reuse system by law. However, only less than 0.5% of the total buildings have more than 10,000 m2. Therefore, the regulation is ineffective and merely nominal. Second, service water is supplied at low charge (0.20 US-dollar/m3 water). The inexpensive service water often discourages people to recycle treated wastewater. Third, people still think recycled water is not clean enough and can cause diseases. Therefore, they should be informed that a well-maintained recycling system does not fail to produce water with high quality.

An expression study of hormone receptors in spontaneously developed, carcinogen-induced and hormone-induced mammary tumors in female Noble rats.

The Noble (Nb) rat model has been used in the study of hormonal carcinogenesis of mammary and prostate glands, as this rat strain is susceptible to tumor induction in these glands by hormonal treatments. Recently, we demonstrated that this rat strain can develop spontaneously mammary tumors at high incidence in aged animals and also show high sensitivity to chemical carcinogens (DMBA and MNU) and combined treatments with sex hormones in mammary tumor induction. In the present study, we examined and compared the expression of hormone receptors [including estrogen receptors (ERalpha and ERbeta), androgen receptor (AR), progesterone receptor (PR), prolactin receptor (PRLR)] and prolactin (PRL) by immunohistochemistry, Western blotting and RT-PCR in spontaneous mammary tumors, and mammary tumors induced by sex hormones (T+E2 and T+DES for 8-10 months) and DMBA in Nb rat model. Immunohistochemistry and Western blotting showed that both the spontaneously developed and hormone-induced carcinomas exhibited strong immunoreactivity of ERalpha, ERbeta, AR, PR and PRLR, while the spontaneous fibroadenomas showed weak to moderate immunoreactivity of ERalpha and PRLR, whereas the DMBA-induced carcinomas exhibited weak to moderate immunoreactivity of ERalpha, AR, PR and PRLR, and sporadic weak ERbeta immunoreactivity. RT-PCR analyses showed that mRNA expression pattern of these markers resembled that of proteins. In addition, weak mRNA expression of PRL was detected in spontaneous carcinomas and carcinomas induced by DMBA and hormones, suggesting that PRL could be produced locally within the tumors. The results showed that the expression status of hormone receptors and PRL was different in spontaneous mammary tumors and tumors induced by carcinogen or hormones, suggesting that the extent of involvement of steroid hormones and their receptors in the spontaneous, carcinogen- or hormone-induced mammary carcinogenesis might be different.

Spontaneous mammary tumors in aging Noble rats.

Noble (Nb) rat strain has been used for the study of hormonal carcinogenesis of mammary and prostate glands, for its susceptibility to develop premalignant lesions as well as carcinomas in these organs by sex hormones. However, background information on the spontaneously developed mammary tumors in this rat strain is scarce. We report on the incidence rate, latency period and histopathology of mammary tumors spontaneously developed in the senile intact and untreated Nb rats compared with those induced by either combined treatments with sex steroids or 7,12-dimethylbenz[a]anthracene (DMBA) in the same rat strain. We observed that the incidence rate of spontaneous mammary tumors was 45% in female Nb rats and 3% in the males. The average age of the female Nb rats to develop palpable tumors was 14 months and rarely detected in animals less than 12 months old. It was also noted that the incidence rate of the spontaneous mammary tumors was similar to those induced by combined treatments with sex steroids for 8-10 months (46.7% for T+E2 and 55.6% for T+DES) but less than those by DMBA treatment in 8 months (over 80%). Histologically, majority of the spontaneous mammary tumors were fibroadenomas, which comprised 70% of all collected tumors and about 20% were carcinomas whereas tumors induced by steroid hormones and DMBA were all carcinomas. Distant metastases of spontaneous mammary carcinomas to lung, liver and lymph nodes were also noted, but rarely.

Expression pattern of glycoconjugates in rat retina as analysed by lectin histochemistry.

The present study sought to characterize the expression and distribution of complex glycoconjugates in the rat retina by lectin histochemistry, using a panel of 21 different lectins with different carbohydrate specificities. Paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with various biotinylated lectins, followed by the streptavidin-peroxidase and glucose oxidase-diaminobenzidine-nickel staining procedures. The results showed that the retinal pigment epithelium was stained intensely with LCA, Jacalin, WFA, S-WGA, PWA, DSA, UEA-I, LTA and PHA-E, suggesting that this epithelium contained glycoconjugates with alpha-Man, alpha-Glc, alpha-Gal/GalNAc, beta-GalNAc, alpha-Fuc, NeuAc and other oligosaccharide residues. The outer and inner segments of the photoreceptor layer showed different lectin binding affinities. The outer segments reacted with S-WGA and GS-II, whereas the inner segments reacted with UEA-II, UEA-I, LTA and MAA, suggesting that the inner segments contained glycoconjugates rich in alpha-Fuc and NeuAc(alpha2,3)Gal residues. PNA labelled specifically the cones and could be used as a specific marker for these photoreceptors. RCA-I, WFA, S-WGA, DSA, MAA and PHA-E reacted with both the outer and inner plexiform layers. On the other hand, UEA-I and LTA specifically labelled the outer plexiform layer, while PNA labelled the inner plexiform layer. The retinal microglial cells were labelled specifically by GS-I-B4 and SNA. Interestingly, we also observed that WFA bound specifically to Müller cells and could be used as a novel marker for this retinal glial cell. The capillaries and larger vessels in the retina and choriocapillaris reacted intensely with GS-I-B4, RCA-I, S-WGA, PWA, DSA and PHA-E. No significant differences in lectin binding were observed in the microvessels at these two sites. In summary, the present study demonstrated the expression patterns of glycoconjugates in the rat retina and that certain lectins could be used as histochemical markers for specific structural and cellular components of the rat retina.

Altered expression of extracellular matrix and proteinases in Noble rat prostate gland after long-term treatment with sex steroids.

BACKGROUND: Interactions between the epithelial cells and stromal tissues, which include the epithelial basement membrane, extracellular matrix, inducible factors, and various cell types, are believed to play a significant role in prostate gland carcinogenesis. Remodeling of extracellular matrix and degradation of basement membrane are the prerequisites for tumor cell invasion, and these changes are correlated with the expression of various proteinases. METHODS: The present study examined the alterations of epithelial basement membrane, extracellular matrix, and proteinase activities in the Noble rat prostate gland after long-term treatments with androgen and estrogen (T+DES or T+E(2) for 4-12 months) by histochemistry, immunohistochemistry, electron microscopy, and gelatin-gel zymography. RESULTS: After hormonal treatments, defects of epithelial basement membranes, such as focal disruption, diffuse staining and multilayering, were observed by histochemistry and immunohistochemistry in the dysplastic and neoplastic lesions induced in the lateral (LP) and ventral prostates (VP) but not in dorsal prostate (DP). An increase in the amount of extracellular matrix components, including hyaluronan (HA), chondroitin sulfate proteoglycan (CSPG) and tenascin, in the stroma of hormone-treated LP and VP was revealed by histochemistry and immunohistochemistry. Positive immunolabeling of matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) was detected in the fibromuscular layer surrounding the adenoma and adenocarcinoma induced in LP and VP after treatments with steroids for over 9-12 months. Zymography also detected an increase in activities of proteinases of apparent MW 120, 90, 86 and 68 kDa in the hormone-treated LP and VP, and these proteinases were characterized as metalloproteinases. In addition, two serine proteinases of MW 100 and 30 kDa were identified as being overexpressed in the hormone-treated LP and VP. Compared to LP and VP, there was no significant change in the proteinase activities in the hormone-treated DP. CONCLUSIONS: The present study demonstrated that the epithelial basement membrane and stromal extracellular matrix were altered in dysplastic and neoplastic Noble rat prostates. Since HA and CSPG (or their complexes) are highly anionic molecules, their increased accumulation in the altered prostatic stroma would tend to hydrate this tissue. This would create an environment more favorable for tumor growth and invasion. These morphological changes were also correlated with the concurrent increase in gelatinolytic proteinase activities induced in these prostates. The results suggest that the remodeling of the stromal tissue might play a role in the early stage of prostate carcinogenesis as shown in the Noble rat model.

Analysis of glycoconjugate patterns of normal and hormone-induced dysplastic Noble rat prostates, and an androgen-independent Noble rat prostate tumor, by lectin histochemistry and protein blotting.

BACKGROUND: Alteration of the expression of glycoconjugates is frequently observed in tumors. However, studies on the changes of cellular glycosylation in the early premalignant stage of prostate carcinogenesis are scarce. METHODS: The present study characterized and compared the glycoconjugates expressed in the dysplastic lateral prostate induced in Noble (Nb) rat by steroid hormones and a transplantable androgen-independent Nb rat prostatic carcinoma line (AIT) by lectin histochemistry and protein blotting. RESULTS: The results of lectin histochemistry show that the dysplastic prostatic epithelium elaborates altered patterns of glycosylation, which are distinct from the normal secretory epithelium. Some individual cells in the dysplastic epithelium were intensely labeled by the N-acetylgalactosamine (GalNAc)-specific (agglutins from Glycine max [SBA], Helix aspera [HAA], Helix pomatia [HPA], Vicia villosa [VVA], Erythrina cristigalli [ECA]) and complex-type oligosaccharide-specific (Phaseolus vulgaris agglutin [PHA-E]) lectins, indicating that these cells contained abundant GalNAc(alpha1,3)GalNAc/Gal and Gal(beta1,4)GlcNAc(alpha1,2)Man(alpha1,6) residues. These lectins also bound to some tumor cells in the AIT, suggesting that these sugar residues are common in some dysplastic and neoplastic prostatic cells. The study has also identified several lectins (agglutins from Griffonia simplicifolia [GS-I-B4], Arachis hypogaea [PNA], Ricinus communis [RCA-I], Maackia amurensis [MAA], Sambucus nigra [SNA]), which bound only to some AIT tumor cells but not to dysplastic epithelium, indicating that alpha/betaGal and sialic acid-containing glycoconjugates are expressed by neoplastic prostatic cells. The results of lectin blottings with Triticum vulgare agglutin [S-WGA] Ulex europaeus agglutin [UEA-I] and PHA-E have identified five major glycoprotein bands (of apparent molecular weights of 116, 79, 64, 61, and 57 kDa) in the microsomal fraction of testosterone plus 17beta-estradiol (T + E2)-treated lateral prostate. These lectin-reactive bands were not detected in the AIT extracts. In the AIT microsomal extract, two glycoprotein bands of molecular weights of 58 and 46 kDa were revealed by SBA and PNA. CONCLUSIONS: The present study shows that there is an increased expression of GalNAc(alpha1,3)GalNAc/Gal residues and triantennary complex-type oligosaccharides in the dysplastic epithelial cells as compared to normal secretory epithelial cells in rat lateral prostate. This altered expression of glycoconjugates revealed in the dysplastic epithelium indicates an aberrant glycosylation in the early premalignant stage of prostate carcinogenesis. The results also show that the AIT tumor cells are heterogeneous in their glycoconjugates and different from the dysplastic epithelial cells.

Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin.

The flavonoid baicalin (baicalein 7-D-beta-glucuronate), isolated from the dried root of Scutellaria baicalensis Georgi (Huang Qin), is widely used in the traditional Chinese herbal medicine for its anti-inflammatory, anti-pyretic and anti-hypersensitivity effects. In the present study, we investigated the in vitro effects of baicalin on the growth, viability, and induction of apoptosis in several human prostate cancer cell lines, including DU145, PC-3, LNCaP and CA-HPV-10. The cell viability after treating with baicalin for 2-4 days was quantified by a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium (MTS) assay. The results showed that baicalin could inhibit the proliferation of prostate cancer cells. The responses to baicalin were different among different cell lines, with DU145 cells being the most sensitive and LNCaP cells the most resistant. Baicalin caused a 50% inhibition of DU145 cells at concentrations of 150 microM or above. The inhibition of proliferation of prostate cancer cells after a short period of exposure to baicalin was associated with induction by apoptosis, as evidenced by the typical nuclear fragmentation using Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling, DNA fragmentation, activation of caspase-3 and cleavage of poly-ADP-ribose polymerase (PARP). The results indicate that baicalin has direct anti-tumor effects on human prostate cancer cells.

PSP94 (or beta-microseminoprotein) is a secretory protein specifically expressed and synthesized in the lateral lobe of the rat prostate .

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94), also called beta-microseminoprotein, is a small, nonglycosylated protein, rich in cysteine residues. It was first isolated as a major protein from human seminal plasma. Subsequently, its homologous proteins were identified, and their cDNAs or genes have been cloned in primates, pigs, and rodents. METHODS: The present study investigated the expression pattern of PSP94 in the normal Noble rat prostate gland by nonradioactive in situ hybridization, Northern blotting, RT-PCR, Western blotting, and immunohistochemistry. Its expression in the mouse prostate gland was also examined by in situ hybridization. RESULTS: The results of in situ hybridization, and Northern and Western blot analyses, showed that the expression of rat PSP94 was prostate-specific. It was highly expressed in the lateral prostatic lobe, moderate in the dorsal lobe, weak in the coagulating gland, and negative in the ventral lobe and seminal vesicle. Its specific expression in the rat prostate gland was further confirmed by RT-PCR analysis of prostatic and nonprostatic organ tissues. Its mRNA transcripts were not detected in the urinary, digestive, and respiratory tracts, male and female reproductive organs, muscles, brain, and kidney. Its molecular mass was estimated to be 14.5 kDa by Western blotting. Similar prostate-specific expression of PSP94 was also observed by in situ hybridization in the lateral lobe, but not in the dorsal and ventral lobe, of the mouse prostate gland. CONCLUSIONS: Rat PSP94 is a major secretory protein highly expressed and synthesized by the lateral lobe of both rat and mouse prostate glands, and moderately expressed in the dorsal lobe of the rat prostate gland.

Ultrastructural and biochemical observations on the early changes in apoptotic epithelial cells of the rat prostate induced by castration.

The present study describes the sequential ultrastructural changes in the apoptotic cells of the rat ventral and dorsal prostates during the early period of 1-3 days postcastration. The major morphological changes include: (1) condensation of heterochromatin along the nuclear envelope and fragmentation into crescent-shaped micronuclei; (2) formation of membrane-bound cytoplasmic spherical bodies, which contain various organelles and micronuclei, within the apoptotic cells; (3) formation of non-membrane-bound autolytic vacuoles by autolysis of cytoplasm; (4) focal rupture of outer mitochondrial membrane; and (5) phagocytosis of the fragmented cytoplasmic spherical bodies and apoptotic cells by macrophages. The occurrence of both cytoplasmic apoptotic bodies and autolytic vacuoles in apoptotic cells suggests that the cytoplasm of the apoptotic cells could be destroyed by different means. The responsiveness of different prostatic lobes to androgen withdrawal and the time course of the transitory apoptotic activity in different lobes were analyzed by counting the indices of the TUNEL-labeled apoptotic cells against the postcastration periods. The results showed that the ventral lobe responded more rapidly to castration than the lateral and dorsal lobes. The dorsal lobe was the slowest in response to castration among the three lobes. Analysis of protease activities by zymography has identified two Ca(2+)-independent proteases of apparent MW 20 and 24 kDa (expressed in both ventral and dorsolateral lobes), and one Ca(2+)-dependent protease of MW 66.5 kDa (expressed only in the dorsolateral lobe) which became activated at day 3 postcastration. Their expression patterns were different from that of CPP-3 in the castrated prostates, suggesting that the activated proteases were enzymes other than CPP-3. The association of their highest activities with the maximum apoptotic activity at day 3 postcastration and also their loss of activity at day 15 suggest that these protease activities might be related to apoptosis or glandular involution.

In situ hybridization study of PSP94 (prostatic secretory protein of 94 amino acids) expression in human prostates.

BACKGROUND: The prostatic secretory protein of 94 amino acids (PSP94), also named beta-microseminoprotein, is one of the major proteins secreted by the human prostate. However, its value as a prognostic marker for prostate cancers is still under debate. The aim of the present study was to examine the expression pattern of this protein in fetal, pubertal, and aged human prostates. METHODS: Nonisotopic in situ hybridization using a digoxigenin-labeled riboprobe for PSP94 and immunohistochemistry were used to demonstrate the expression of PSP94 in different regions or zones of fetal, pubertal, and adult human prostates. Its localization pattern was also compared with those of two other major secretory proteins, prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP), by immunohistochemistry. RESULTS: PSP94 mRNA and its protein were localized to the secretory epithelium of normal pubertal and adult human prostates. No hybridization signal and immunoreactivity of PSP94 were seen in fetal prostates at 6-7 months of gestation, whereas some glandular cells were positive to PSA and PAP immunostainings. In the adult prostates, PSP94 expression was intense in the acini in the peripheral zone, less intense in the transition zone, and variable in the central zone. Such a zonal expression pattern was more apparent in the pubertal prostates. However, no obvious differential expression pattern was observed in the immunohistochemistry of PAP and PSA, which showed a uniform staining of the secretory epithelia of the acini in all anatomic zones. The hybridization signals and immunoreactivity of PSP94 became reduced or lost in premalignant prostatic intraepithelial neoplastic lesions and different grades of prostatic carcinomas. CONCLUSIONS: Fetal prostates at 6-7 months of gestation already synthesize PSA and PAP but not PSP94. The delayed expression of PSP94 appears to correlate with the development of the prostate gland. A differential expression pattern of PSP94 is demonstrated in different anatomical zones, showing that this protein is more expressed and synthesized in the acini in the peripheral zone than in the central and transition zones. However, such a zonal pattern is not seen in the immunohistochemistry of PSA and PAP. The present study also shows that PSP94 is downregulated in different grades of prostate cancers.

Glycoconjugates of the rat ciliary body epithelium: a lectin histochemical and protein blotting study.

The present study sought to identify and partially characterize the glycoconjugates specific to the double-layered ciliary body epithelium of the rat eye by lectin histochemistry and lectin blottings. Hydrated paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with a panel of 21 different biotinylated lectins, followed by streptavidin-peroxidase and the glucose oxidase-diaminobenzidine-nickel staining procedure. The results of lectin histochemistry revealed that the inner epithelial layer was rich in GlcNAc(beta1,4)GlcNAc, alpha-Gal, Gal(beta1,3)GalNAc, GalNAc(alpha1,3)GalNAc/Gal, GalNAc(alpha1,6)Gal, Fuc(alpha1,2)Gal(beta1,4)GlcNAc and Gal(beta1,4)GlcNAc(beta1,2)Man(alpha1,6) sugar residues as shown by its positive reactivities with S-WGA, PWA, DSA, GS-I-B4, PNA, DBA, SBA, WFA, UEA-I, LTA and PHA-E. The reactivities of GS-I-B4, PNA, DBA and SBA were restricted to the inner layer at the tips of the ciliary processes. On the other hand, the outer epithelial layer was stained evenly by DSA and Jacalin, and partly by MAA, showing that this epithelial layer was rich in GlcNAc(beta1,4)GlcNAc, Gal(beta1,3)GalNAc and NeuAc(alpha2,3)Gal disaccharides. These lectin binding patterns of the ciliary body epithelium suggest a topographical and functional difference in this double cell-layered epithelium. Their possible roles in the secretion of aqueous humour and production of ciliary zonule are discussed. Some identified lectin markers specific to these two cell layers may be useful for further experimental studies. Glycoproteins extracted from the dissected ciliary body were separated by SDS-PAGE electrophoresis and analyzed by protein blottings with 8 different lectins. The results showed that at least 10 major membrane-bound glycoproteins, with molecular weights ranging from 30 to 150 kD, rich in beta-GlcNAc, beta-Gal, alpha/beta-GalNAc and NeuAc(alpha2,6)Gal residues, were present in the microsomal fraction.

Hyaluronan and chondroitin sulfate proteoglycans are colocalized to the ciliary zonule of the rat eye: a histochemical and immunocytochemical study.

In previous studies, chondroitin sulfate proteoglycans have been localized to the periphery of the zonular fibers and the individual zonular fibrils (or microfibrils) after Cuprolinic blue staining in conjunction with chondroitinase digestions and immunogold labelling with 2-B-6 antibody. In the present study, we wished to determine if these proteoglycans are linked to hyaluronan to form a large multimolecular aggregate. To accomplish this, we localized the hyaluronan using a biotinylated hyaluronan-binding protein fragment of chondroitin sulfate proteoglycan, containing also the link protein, purified from bovine nasal cartilage. The results showed that the ciliary zonule of the rat eye was reactive with the biotinylated hyaluronan-binding probe as demonstrated by streptavidin-peroxidase-diaminobenzidine staining and streptavidin-gold labelling. Hyaluronan-gold labelling showed that the gold particles were mostly localized on the periphery of the zonular fibers, which was similar to the localization pattern of the zonule associated-proteoglycans. This hyaluronan-binding probe also strongly labelled the sites of zonule insertion over the basement membrane of the inner ciliary epithelium at the pars plana and the lens capsule at the equatorial region, which suggests its probable role in the attachment of ciliary zonule to the basement membranes. To demonstrate whether these two molecules are linked to one another, ultrastructural colocalization of both hyaluronan and chondroitin sulfate proteoglycans was performed on the same sections by double-gold labelling, and combined Cuprolinic blue staining and hyaluronan-gold labelling. Gold particles of 15 and 10 nm in sizes labelling both hyaluronan and chondroitin 4-sulfate, were colocalized to the surface of the zonular fibers. The combined Cuprolinic blue staining and hyaluronan-gold labelling showed that the gold particles were localized towards the ends of the Cuprolinic blue-stained rodlets, which strongly suggests that these chondroitin sulfate proteoglycans are linked to the hyaluronan chain to form a large aggregate surrounding the periphery of the zonular fibers. These ciliary zonule-associated proteoglycan-hyaluronan aggregates may play a role in organizing the individual zonular fibrils (microfibrils) into bundles of zonular fibers.

Proteoglycans associated with the ciliary zonule of the rat eye: a histochemical and immunocytochemical study.

The structural organization of integral and associated components of the ciliary zonule is still not fully understood. The present study is to localize and characterize the proteoglycans associated with the ciliary zonule of the rat eye by Cuprolinic blue (CB) staining and immunocytochemistry. After CB staining, the proteoglycans appeared as electron dense elongated rodlets and were localized with the zonular fibers. They were seen lying on the periphery of the zonular fibers or along the length of the individual fibrils. Most of the CB rodlets had a size of 60-170 nm long (average 130 nm) and 25 nm wide. Smaller CB rodlets measuring 25-60 nm long (average 45 nm) and 12 nm wide were sometimes found associated with the individual zonular fibrils. The CB rodlets were removed after chondroitinase ABC or chondroitinase AC treatment, but were resistant to heparitinase, nitrous acid, keratanase or Streptomyces hyaluronidase digestions. The ciliary zonule was also immunostained with three monoclonal antibodies: 2-B-6 specific for chondroitin 4-sulfate, 3-B-3 for chondroitin 6-sulfate and 1-B-5 for unsulfated chondroitin, using indirect immunoperoxidase or immuno-colloidal gold methods. The zonular fibers were immunoperoxidase stained and immunogold labeled by 2-B-6, but were not reactive to 3-B-3 and 1-B-5. The results demonstrate that chondroitin sulfate proteoglycan is associated with the ciliary zonule of the rat eye.

Prenatal cocaine exposure revealed minimal postnatal changes in rat striatal dopamine D2 receptor sites and mRNA levels in the offspring.

It has been reported from this laboratory that prenatal cocaine exposure results in the postnatal transient alterations of rat striatal dopamine uptake sites examined from postnatal 0-32 wk. The present study aims to examine whether this will result in a direct/indirect stimulation of dopamine D2 receptors. Pregnant rats were dosed orally with cocaine hydrochloride (60 mg/kg/d) from gestational day (GD) 7-21. Control animals received an equivalent volume of water. The striatum from the offspring at postnatal 0-32 wk was examined. The radioligand [3H]sulpiride was used for the Scatchard analysis of the D2 receptors, and the changes in the levels of mRNA for the D2 receptor were studied using Northern blot analysis. Results from the present study revealed that in the control group, there was an age-dependent increase in the number of D2 receptor sites (Bmax: 44.00 +/- 2.12 to 178.00 +/- 45.10 fmol/mg protein) and in the levels of D2 mRNA from PN0-32 wk with the most rapid increase occurring during the first 4 wk of postnatal development. Prenatal cocaine exposure resulting in only a significant decrease (p < 0.001) in the number of D2 receptor sites at PN0 wk and in a 10% increase in mRNA levels at PN3, 4, and 12 wk. It was concluded from this study that prenatal cocaine exposure resulted in minimal postnatal changes in the dopamine D2 receptor.

Postnatal changes in [3H]mazindol-labelled dopamine uptake sites in the rat striatum following prenatal cocaine exposure.

The present study showed that prenatal cocaine exposure (60 mg/kg/day) has a transient effect on the [3H]mazindol-labelled dopamine uptake sites in the striatum of the rat offspring examined from postnatal week 0-32. There is a 39% and 21% decrease in the number of binding sites (Bmax) in the cocaine-exposed group at postnatal weeks 3 and 4, respectively, with a recovery to near normal values by postnatal week 8.