Prenatal morphine alters the synaptic complex of postsynaptic density 95 with N-methyl-D-aspartate receptor subunit in hippocampal CA1 subregion of rat offspring leading to long-term cognitive deficits.

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

Paracrine function of cultured aortic endothelial cells in spontaneously hypertensive rats.

BACKGROUND: Vasoconstrictor and vasodilator release from vascular endothelial cells not only regulates vascular tone but also induces vascular smooth muscle cell proliferation. METHODS: In order to understand the role of vasoconstrictor and vasodilator release in the development of hypertension in spontaneously hypertensive rats (SHR), aortic endothelial cells were isolated and cultured from 4-week-old and 24-week-old SHR (SHR-4 and SHR-24) and age-matched Wistar-Kyoto rats (WKY-4 and WKY-24) used as control. Prostacyclin (PGI2), endothelin-1 (ET-1) and thromboxane A2 (TXA2) release from cultured endothelial cells in the culture medium, were measured after 30 min with or without treatment with acetylcholine, calcium ionophore A23187 or thrombin. RESULTS: The results showed that there was no significant difference in ET-1 secretion between SHR-4 and age-matched WKY rats, but ET-1 secretion was about twice as high in SHR-24 as in WKY-24. TXA2 secretion was significantly higher in SHR-4 than in WKY-4 and was also higher than in SHR-24, but there was no significant difference between SHR-24 and WKY-24. The secretion of PGI2 was higher in SHR-24 than in WKY-24 and also higher than in SHR-4 and WKY-4. The prostaglandin PGI2 and TXB2 secretions from all groups of cultured VECs treated with various reagents, acetylcholine, calcium ionophore A23187 or thrombin were increased in similar patterns. However, there was no significantly different response between SHR and WKY VECs. CONCLUSIONS: Similar levels of ET-1 secreted from endothelial cells between SHR-4 and WKY-4 indicated that ET-1 secretion seems not a crucial factor in early hypertension development in SHR. The high level of TXA2 secretion in SHR-4 may involve in early hypertension development in SHR.

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte.

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.

GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation.

Surface glycoconjugates of spermatozoa are modified during epididymal maturation, which is closely related to the development of sperm function. In addition, recognition of surface glycoconjugates is one of very critical events in sperm-oocyte interaction. The binding of carbohydrate-specific lectins to the human sperm surface during epididymal maturation has been investigated. However, the glycoproteins responsible for lectin binding in sperm maturation are not well documented. This study used wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (Con-A) to identify sperm maturation-related glycoproteins in human epididymis. Histochemical localization revealed that the binding sites of WGA, PNA and Con-A were mainly in the principal cells and luminal contents of the human epididymis, but not in the interstitial regions. Each lectin displayed a fairly distinct regional localization. On Western blots probed with WGA and Con-A, glycoproteins of 83 kDa (GP-83) and 39 kDa (GP-39) were identified in the sperm extracts, epididymal fluid and tissue extracts of the corpus and cauda epididymides, but not in the caput. PNA identified GP-83 in the same manner as WGA and Con-A, but did not recognize GP-39. These results suggest that lectin-binding glycoproteins GP-83 and GP-39 found on mature spermatozoa may be secreted by the principal cells of corpus and cauda epididymis, and conjugated to spermatozoa during their transit in human epididymis.

Isolation and characterization of proteins that bind to galactose, lipopolysaccharide of Escherichia coli, and protein A of Staphylococcus aureus from the hemolymph of Tachypleus tridentatus.

In this study, we report the isolation and characterization of three novel hemolymph proteins that are believed to be involved in the innate immune response of horseshoe crabs, Tachypleus tridentatus. They include two closely related proteins, one that binds to the protein A of Staphylococcus aureus (PAP) and another that binds to the lipopolysaccharide of Escherichia coli (LBP). PAP binds specifically to staphylococcal protein A (SpA) with a K(D) of 3.86 x 10(-5) M, whereas LBP binds to lipopolysaccharide (LPS) with a K(D) of 1.03 x 10(-6) M. Both PAP and LBP are glycoproteins with an apparent molecular mass of about 40 kDa. N-terminal sequences of PAP and LBP showed 61.9 and 72.2% identity, respectively, to tachylectin-3, a lectin isolated from the amebocyte of T. tridentatus, previously characterized by its affinity to the O-antigen of LPS and blood group A antigen (Muta, T., and Iwanaga, S. (1996) Curr. Opin. Immunol. 8, 41-47). The third protein, a galactose-binding protein (GBP), was found to bind tightly to Sepharose CL-4B and could only be eluted from the column matrix with chaotropic agents, such as 4 M urea or 2 M guanidine hydrochloride. Further analysis indicated that GBP binds to D(+)-galactose with a K(D) of 2.47 x 10(-7) M. N-terminal sequence analysis showed that GBP shared a 50% identity with lectin L-6, identified in the granules of amebocyte of T. tridentatus. (Gokudan, S., Muta, T., Tsuda, R., Koori, K., Kawahara, T., Seki, N., Mizunoe, Y., Wai, S. N. , Iwanaga, S., and Kawabata, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10086-10091). Lectin-L6 and tachylectin-3 are nonglycosylated intracellular proteins with about half the molecular mass of PAP, LBP, and GBP. GBP also binds to PAP and LBP with K(D) values of 1.25 x 10(-7) and 1.43 x 10(-8) M, respectively, and this binding is enhanced about 10-fold upon the addition of SpA and LPS to form the GBP.PAP.SpA and GBP.LBP.LPS complexes, respectively.

On the biosynthesis of alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12.

Escherichia coli Bos-12 synthesizes a heteropolymer of sialic acids with alternating alpha-2,9/alpha-2,8 glycosidic linkages (1). In this study, we have shown that the polysialyltransferase of the E. coli Bos-12 recognizes an alpha-2,8 glycosidic linkage of sialic acid at the nonreducing end of an exogenous acceptor of either the alpha-2,8 homopolymer of sialic acid or the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid and catalyzes the transfer of Neu5Ac from CMP-Neu5Ac to this residue. When the exogenous acceptor is an alpha-2,8-linked oligomer of sialic acid, the main product synthesized is derived from the addition of a single residue of [14C]Neu5Ac to form either an alpha-2,8 glycosidic linkage or an alpha-2,9 glycosidic linkage at the nonreducing end, at an alpha-2, 8/alpha-2,9 ratio of approximately 2:1. When the acceptor is the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid, chain elongation takes place four to five times more efficiently than the alpha-2,8-linked homopolymer of sialic acid as an acceptor. It was found that the alpha-2,9-linked homopolymer of sialic acid and the alpha-2,8/alpha-2,9-linked hetero-oligomer of sialic acid with alpha-2,9 at the nonreducing end not only failed to serve as an acceptor for the E. coli Bos-12 polysialyltransferase for the transfer of [14C]Neu5Ac, but they inhibited the de novo synthesis of polysialic acid catalyzed by this enzyme. The results obtained in this study favor the proposal that the biosynthesis of the alpha-2, 9/alpha-2,8 heteropolymer of sialic acid catalyzed by the E. coli Bos-12 polysialyltransferase involves a successive transfer of a preformed alpha-2,8-linked dimer of sialic acid at the nonreducing terminus of the acceptor to form an alpha-2,9 glycosidic linkage between the incoming dimer and the acceptor. The glycosidic linkage at the nonreducing end of the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid produced by E. coli Bos-12 should be an alpha-2,8 glycosidic bond and not an alpha-2,9 glycosidic linkage.

Frequent aberration of the transforming growth factor-beta receptor II gene in cell lines but no apparent mutation in pre-invasive and invasive carcinomas of the uterine cervix.

The type II transforming growth factor-beta (TGF-beta) receptor (RII) gene located at 3p22 plays an important role in regulating growth and differentiation of epithelium, including that of the uterine cervix. Loss-of-function mutations of RII have frequently been found in gastrointestinal cancers, with a replication-error (RER) phenotype characterized by the presence of microsatellite instability (MI). In this study, genomic PCR, SSCP and DNA sequencing were conducted to investigate the coding sequences of the RII gene in cell lines (n = 5) and tissues (n = 15) of squamous carcinomas of the uterine cervix. Intragenic deletions were noted in 2 of 5 cervical-cancer cell lines (ME180 and HeLa cells). However, no mutation, other than DNA polymorphisms, was found in 15 cervical cancers with either alleleic loss at 3p22 (n = 11) or MI (n = 4). Further analysis of squamous intraepithelial lesions (SIL) with (n = 12) or without (n = 4) MI for the (A)10 change, a prototypic mutation found in over 90% of RER-positive colon cancers, also showed no aberration. Our study concludes that the RII gene is frequently disrupted in cervical-cancer cell lines, but is rarely mutated in CC and SIL tissues, including those showing MI or alleleic loss at 3p22. The underlined mechanism of genomic instability in CC and SIL may thus differ from that of colorectal cancer. The allelic loss at 3p22-24 in CC does not involve the coding sequence of the RII gene. The non-coding sequence of RII or an unidentified gene may be responsible for it.

Thromboxane and prostacyclin in maternal and fetal circulation in pre-eclampsia.

OBJECTIVES: A major pathophysiologic change of pre-eclampsia has been attributed to the overproduction of thromboxane A2 (TXA2) mainly from activated platelets. On the other hand, increased biosynthesis of TXA2 has also been reported from preeclamptic placentas. The systemic role of these different sources of TXA2 has not been clarified. The purpose of this study is to define the changes of TXA2 and the antagonizing prostacyclin (PC) in maternal and fetal circulations. METHODS: The stable metabolites of TXA2 and PC [Thromboxine B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), respectively] in the cord and maternal blood of nine patients with pre-eclampsia and nine normal parturients were measured by radioimmunoassay. RESULT: In normal pregnancy, the cord blood contained much higher TXB2 (1697+/-898 vs. 267+/-128 ng/ml, P < 0.01) and 6-keto-PGF1alpha (266+/-263 vs. 12.5+/-3.9 ng/ml, P < 0.05) levels than the maternal blood. In the preeclamptic state, a marked increase of TXB2 was noted in both maternal and cord blood, reaching levels which were significantly higher than during normal pregnancy (2995+/-1103 vs. 267+/-128 ng/ml in maternal blood, P < 0.0001, and 3197+/-1288 vs. 1697+/-898 ng/ml in cord blood, P < 0.005). A less significant increase in 6-keto-PGF1alpha (134+/-10.8 vs. 12.5+/-3.9 ng/ml, P < 0.05) was also noted in the maternal blood. Moreover, the level of TXB2 correlated with the diastolic blood pressure of preeclamptic patients before and after delivery. CONCLUSION: The results suggest an abundant source of eicosanoids in the feto-placental circulation, which does not readily cross the placental barrier. In pregnancy complicated with pre-eclampsia, thromboxane level of both fetal and maternal circulations are markedly increased, which may be responsible for the pathophysiologic changes. The lack of adverse systemic effects on the fetus highlights a placental source of TXA2 of transient bioactivity which is rapidly hydrolyzed to non-active TXB2. Federation of Gynecology and Obstetrics

The correlation of blood pressure, age and endothelin-1 binding sites in aortic smooth muscle cells of rats.

Spontaneously hypertensive (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD) rats at the ages of 4, 8, 12, 16 and 24 weeks were used to examine the effects of age on the density of endothelin-1 (ET-1) binding sites in aortic smooth muscle cells and systolic blood pressure (SBP). The SBP of the 3 different rat strains was measured, and the maximum binding value (Bmax) and dissociation constant (Kd) of ET-1 binding sites in smooth muscle cells of the thoracic aorta were determined. The results showed that the SBP and Bmax values of SHR, WKY and SD rats increased with age; the SBP and Bmax value at each corresponding age were significantly higher in SHR than in WKY and SD rats, however, there was no significant difference between WKY and SD rats. The relationship of age vs SBP, age vs Bmax, and Bmax vs SBP showed significantly positive correlation in all 3 rat strains. The regression line in the Bmax of endothelin binding sites against SBP in the 3 different rat strains presented a similar slope. These results indicate that SBP, which increased with age, could be related to an increased density of ET-1 binding sites on vascular smooth muscle cells in these 3 different rat strains.

Comparison of endothelin binding sites in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive rats.

Endothelin-1 (ET-1), which is secreted from vascular endothelial cells, is not only a potent vasoconstrictor but also a vascular smooth muscle cell growth factor. The direct effect of ET-1 on vascular smooth muscle cells, mediated via its specific receptor may therefore play an important role in hypertension and atherosclerosis. Our previous studies indicated that ET-1 secretion from cultured aortic endothelial cells from spontaneously hypertensive rats (SHRs) at the prehypertensive stage (4 weeks old) was not significantly different from that of cells from age-matched Wistar-Kyoto (WKY) rats. In this study, the binding of ET-1 to cultured aortic smooth muscle cells from SHRs and WKY rats was studied. Using tissue explant techniques, rat aortic smooth muscle cells from SHRs and age-matched WKY rats of different ages (4 and 24 weeks old) were successfully cultured in vitro. The maximum binding capacity (Bmax) and binding affinity (Kd) of ET-1 to cultured aortic smooth muscle cells were evaluated by a receptor-binding assay. The data revealed that the affinity of ET-1 binding to smooth muscle cells was similar in all 4 groups of experimental rats. However, the Bmax of cultured smooth muscle cells from 24-week-old SHRs was 2.5 times higher than of smooth muscle cells from age-matched WKY rats (8.64 +/- 0.72 vs 3.69 +/- 0.10 fmol/10(6) cells) and 1.5 times higher than in aortic smooth muscle cells from 4-week-old SHRs (8.64 +/- 0.72 vs 5.36 +/- 0.36 fmol/10(6) cells). These results suggest that hypertension in SHRs may be related to a high density of ET-1 receptors on arterial smooth muscle cells.

Enhanced malignant progression of nasopharyngeal carcinoma cells mediated by the expression of Epstein-Barr nuclear antigen 1 in vivo.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) and mostly classified as poorly differentiated squamous cell carcinoma or undifferentiated carcinoma with early metastasis and a rapidly progressive clinical course. The EBV-encoded latent proteins, Epstein-Barr nuclear antigen 1 (EBNA 1) and latent membrane proteins (LMPs), may be expressed in NPC, but their biological effects are poorly understood. EBNA 1 may predispose B lymphocytes to lymphomagenesis in transgenic mice, but its biological effects in NPC are still unknown. This study investigated the biological effects of EBNA 1 by expressing it in an EBV-negative NPC cell line (HONE-1), which was then inoculated into both nude and severe combined immunodeficiency mice. The EBNA 1 caused HONE-1 cells to grow in a less differentiated pattern and to progress more rapidly, as well as increasing their tumourigenicity and metastatic capability. These data suggest that EBNA 1 may play a critical role in the progressive evolution of NPC.

The porcine sperm motility inhibitor is identical to beta-microseminoprotein and is a competitive inhibitor of Na+,K(+)-ATPase.

We have isolated a small porcine seminal protein called SMI-1. Our results from peptide sequence, amino acid composition and mass spectral analyses reveal that SMI-1 is identical to porcine beta-microseminoprotein, a protein with unknown function. We also report here that this protein inhibits competitively the activity of Na+,K(+)-ATPase purified from porcine cerebral cortex in a dose-dependent manner. The inhibitory effect could be reversed by the addition of ATP. The half-maximal inhibition was achieved at an inhibitor concentration of 90 microM.

Isolation and cultivation of aortic endothelial cells from spontaneously hypertensive rat with a modified tissue explant technique.

In the present study, we successfully established a "tissue explant technique" to obtain high yield and purity of endothelial cells from the aorta of hypertensive and normotensive rats (SHR and WKY). Small pieces of aorta were placed on fibronectin precoated petri dishes. The effects of oxygenation in the tissue preparation stage, tilting of the petri dish during the explanting period and timing of the removal of tissue blocks from petri dishes were evaluated. These procedures appeared to be critical for cell survival, tissue adhesion and minimizing of non-endothelial cell contamination. The cultured endothelial cells were characterized by morphological, immunohistochemical and biochemical examination. The cultured cells from both SHR and WKY rats showed similar endothelial cell character, positive immunofluorescence staining for the von Willebrand factor, and uptake of acetylated low-density lipoprotein (DiI-ac-LDL). The secretory function of prostacylcin I2 (PGI2), thromboxane A2 and endothelin of cultured endothelial cells was measured. The results showed that the secretion of both PGI2 and endothelin was greater in SHR than in WKY rats, but that there was no difference in thromboxane A2 secretion. Therefore, our "tissue explant technique" can provide high yield and purity of endothelial cells with their specific biological function in vitro. It will permit us to further study the role of endothelial cells in the development of hypertension.

The induction of multidrug resistance in human cervical carcinoma cell lines by estrogenic hormones.

The role of estrogenic hormones on the induction of drug resistance was studied in cervical cancer cell lines, SiHa and Caski. After the cells were inoculated with estradiol (E2) or diethylstilbestrol (DES) in various dosages, the cell survival rates with adriamycin treatment were examined by MTT (3-[4,5-dimethyl-thiazole-2-yl]-2,5-diphenyl-tetrazolium-bromide) test and the intracellular accumulation of adriamycin was evaluated by flow cytometry. In the same condition, the expression of multidrug resistance gene-1 (mdr-1 gene) was detected either by Northern blot hybridization for mdr-1 mRNA or by immunoblot for P-glycoprotein 170. The data in this study indicated that estrogenic hormones had the capacity to induce drug resistance in cervical carcinoma cell lines. When cells were treated with estrogenic hormones and adriamycin simultaneously, the intracellular accumulation of adriamycin declined and corresponded with the drug resistance. Since the expression of the mdr-1 gene induced by E2 or DES results in drug resistance, it is suggested that the mdr-1 gene in SiHa cells may contain the estrogenic responsive element (ERE) in its regulatory region. However, the mechanism of drug resistance induced by estrogenic hormones in Caski cells is different from SiHa cells due to the absence of mdr-1 gene expression. Despite that, this experiment may provide a model to investigate the relationships between estrogenic hormones and drug resistance in other female genital cancers.

Growth and metabolism of cultured bone cells using microcarrier and monolayer techniques.

The traditional in vitro cell culture methods provide bone cells on matrix-coated Petri dishes to grow the cells in a monolayer. This limits the usefulness in situations where there is a need to suspend anchorage dependent cells. To promote the massive production of the cultured cells and to enable the suspension of the cells in a medium, microcarrier cell culture was developed and evaluated. Isolated bone cells from rat calvaria were incubated in a microcarrier culture flask with Cytodex 1. The microcarrier cell morphology was examined by a phase contrast microscope, a scanning electron microscope, and a transmission electron microscope. Cell growth, enzyme markers, and biosynthetic characteristics were examined and compared for microcarrier and monolayer methods. The results indicate that all the characteristics of the bone cells, whether cultured in the Petri dish or microcarrier, were the same. Therefore, the studies of the function and behavior of bone cells still remain useful using the microcarrier system culture.

Establishment and characterization of the transfectable golden hamster embryo fibroblast cell line.

A diploid, continuous cell line, Golden Hamster Embryo Fibroblast-III (GHEF-III), which had been passaged for one year, was established essentially by a 3T3 protocol from primary culture of 14-day-gestation Golden Hamster embryo fibroblast cells. The cultured fibroblast exhibited monolayer growth and had contact inhibition. In morphological identification by light microscope (LM), transmission electron microscope (TEM) and immunofluorescence examination (IF), these multipolar and spindle-shaped cells had a large ovoid nucleus, enriched rER and mitochondria in the cytoplasm. On the other hand, the vimentin presented in the cell with a random network and capped around the nucleus. The results indicated that the cultured GHEF-III cells were fibroblast in origin. The cells were free of bacterial and mycoplasma contamination. The doubling time in GHEF-III was about 15 hours. Chromosomal analysis of GHEF-III presented a diploid stem cell line with a modal number of 44. No evidence of transformation of GHEF-III was shown by properties of contact inhibition, no colony formation in soft agar, and no tumor growth in nude mice. The transformation of GHEF-III after transfection with pT24-C3, an oncogenic plasmid, was shown by the evidence of loss of contact inhibition, growth in low serum medium, colony formation in soft agar and tumor growth in nude mice. In vitro transformation testing of these cells may provide valuable data in studying the role of tumor transforming genes in carcinogenesis. Owing to the genetic stability and less spontaneous transformation, this GHEF-III cell line can be utilized as a source of recipient cells in transfection assay.

Human papillomavirus 16 DNA in NIH3T3 cells transformed by colonic cancer cellular DNA.

Human papillomavirus (HPV) 16 DNA is closely associated with human cancers. It has been identified as an aetiological agent in cervical cancers and, recently, in colonic neoplasms. To further understand the role of HPV 16 DNA in colorectal carcinogenesis, NIH3T3 cells were transformed with high molecular weight DNA from colonic cancer cells and the expression of HPV 16 DNA detected. Both human Alu and HPV 16 DNA sequences were found in the type II foci of CC-M2T cells by Southern blot hybridisation. Additionally, 100% tumorigenicity in nude mice was seen. This study shows the transfection of HPV DNA from colonic cancers into NIH3T3 mouse cells and suggests that HPV type 16 might be associated with the malignant transformation of colonic cells.

The induction and characterization of antiserum against human placenta 17-beta-hydroxysteroid dehydrogenase.

17-beta-Hydroxysteroid dehydrogenase was purified from human placenta. The purification process included the following steps: 50% saturated ammonium sulfate precipitation; heat treatment; affinity column chromatography; and preparative SDS gel electrophoretic elution. This enzyme was also characterized by HPLC gel filtration and two dimensional gel electrophoresis with isoelectrofocusing. The results indicate that the molecular weight of these enzymes in their native condition is around 68 kDa and 34 kDa in SDS PAGE. Therefore, the active form of the enzyme is an identical dimmer in nature. There are three charged isomers as demonstrated by isoelectrofocusing. The antiserum against the 17-beta-HSD was induced by injection of purified human placenta 17-beta-HSD in rabbits. The antiserum was collected and characterized by ELISA, immunohistochemistry, immunoblot and immunoprecipitation tests. The results showed that the antibody titer was over 1:12,800, and specificity against human placenta 17-beta-HSD was also observed.

The secretion of two sperm maturation-related glycoproteins in BALB/c mouse epididymis.

A well-developed Golgi apparatus and rough and smooth endoplasmic reticulum in the principal cells of the mouse epididymis indicate active protein synthesis. Studies have shown that epididymal secretions are essential for sperm maturation. In a previous study, two wheat-germ agglutinin (WGA)-binding glycoproteins, GP-49 and GP-83, were identified on the surface of mature mouse sperm. In this study, synthesis and secretion of these two glycoproteins were investigated. Apparent WGA-binding was found on the stereocilia and in the apical region of principal cells in the corpus and cauda of epididymis. Post-fixation and pre-embedding cytochemical localization revealed that WGA-binding sites were situated in the Golgi apparatus, multivesicular bodies and stereocilia of principal cells. GP-49 and GP-83 were identified in the Nonidet P-40 homogenates of corpus and cauda epididymidis. In the epididymides of which ductuli efferentes had been ligated for more than 4 weeks, no sperm were found in the lumina of epididymal tubules. WGA-binding sites were present in the corpus and cauda; GP-49 and GP-83 were identified in tissue homogenates of the corpus and cauda as well. These findings suggest that GP-49 and GP-83 of mature sperm may be secreted by the principal cells of the corpus and cauda. These two molecules apparently conjugate to sperm whilst sperm transit through the epididymis.