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Chemotherapy 2011 Jun; 57(3)
Evaluation of elution and mechanical properties of two injectable chemotherapeutic bone cements.
Chemotherapeutic bone cements can both stabilize the bone fractures as well as deliver chemotherapy agents directly to the bone metastatic site and adjacent soft tissue tumors. This study evaluated the in vitro elution and flexural properties of Vert... expand abstractebroplastic™ and Confidence Ultra™ bone cements (Depuy Spine Inc., Raynham, Mass., USA) containing methotrexate. In vitro elution was measured by placing bone cement specimens containing 4 different methotrexate amounts in 20 ml saline, and the methotrexate elution was measured at regular intervals for 672 h. The flexural properties of bone cement containing 2 different initial methotrexate amounts after storage in physiological saline were measured using a 3-point bending test. The drug elution rate depended on the initial methotrexate amount added and the type of bone cement used. The relationship between the initial drug amount added and the drug elution rate was not linear. Methotrexate elution decreased the flexural modulus and strength of specimens; this decrease was not proportional to the initial amount of methotrexate added. The results show that bone cements are well suited for use with chemotherapy agents. However, the elution and mechanical properties of each bone cement-drug amount combination should be thoroughly quantified in vitro before using such a combination in a clinical setting. collapse abstract
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Journal of cellular and molecular medicine 2011 Mar;
Atorvastatin activates heme oxygenase-1 at the Stress Response Elements.
Statins are known to inhibit growth of a number of cancer cells, but their mechanism of action is not well established. In this study, PC-3 and MCF-7 cells were used as models to investigate the mechanism of action of atorvastatin, one of the statins... expand abstract. Atorvastatin was found to induce apoptosis in PC-3 cells at a concentration of 1 μM, and in MCF-7 cells at 50 μM. Initial survey of possible pathway using various pathway-specific luciferase reporter assays showed that atorvastatin activated antioxidant response element (ARE), suggesting oxidative stress pathway may play a role in atorvastatin-induced apoptosis in both cell lines. Among the antioxidant response genes, heme oxygenase-1 (HO-1) was significantly up-regulated by atorvastatin. Preincubation of the cells with geranylgeranyl pyrophosphate (GGPP) blocked atorvastatin-induced apoptosis, but not up-regulation of HO-1, suggesting that atorvastatin-induced apoptosis is dependent on GTPase activity and up-regulation of HO-1 gene is not. Six ARE-like elements (designated StRE1 through StRE6) are present in the HO-1 promoter. Atorvastatin was able to activate all of the elements. Since these StRE sites are present in clusters in HO-1 promoter, up-regulation of HO-1 by atorvastatin may involve multiple StRE sites. The role of HO-1 in atorvastatin-induced apoptosis in PC-3 and MCF-7 remains to be studied. collapse abstract
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American journal of medical genetics. Part A 2010 Nov; 152A(12)
Clinical report of microphthalmia and optic nerve coloboma associated with a de novo microdeletion of chromosome 16p11.2.
Anophthalmia and microphthalmia are etiologically and clinically heterogeneous. We present a 13-year-old boy with microphthalmia and multiple anomalies who was evaluated as part of our research into the etiology of microphthalmia. His clinical featur... expand abstractes included left microphthalmia, persistent hyperplastic primary vitreous and posterior coloboma, right posterior pole coloboma, pectus excavatum, mild hypotonia, mild delays in speech and motor development, and an anxiety disorder with social difficulties. Investigations with a chromosome microarray revealed a de novo deletion of chromosome 16p11.2 of approximately 882 kb in size. Deletions of this region of chromosome 16p11.2 are a newly delineated microdeletion syndrome, but this is the first report of microphthalmia and coloboma associated with monosomy for 16p11.2, and emphasizes the clinical variability that can be present with this deletion. This report contributes to the growing knowledge regarding this microdeletion and suggests that rare copy number changes may be a cause of microphthalmia and other eye anomalies. collapse abstract
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Molecular and cellular biochemistry 2008 Nov; 319(1-2)
Brefeldin A activates CHOP promoter at the AARE, ERSE and AP-1 elements.
Brefeldin A induces apoptosis in PC-3 and MCF-7 cells at a concentration of 30 ng/ml. RT-PCR analyses showed up-regulation of CHOP/GADD153 and splicing of XBP-1 mRNA in brefeldin A-treated cells. CHOP promoter-luciferase reporter assays demonstrated ... expand abstractactivation of AARE, ERSE, and AP-1 elements of CHOP promoter by brefeldin A treatment. The activation of these elements was not affected by preincubation of cells with N-acetyl-cysteine (NAC), L: -buthionine-(S,R)-sulfoximine (BSO), and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), suggesting that activation of CHOP promoter by brefeldin A may not involve oxidative stress or JNK signaling pathway. On the other hand, brefeldin A-induced apoptosis was not affected by NAC and BSO pretreatment, but was completely suppressed by JNK inhibitor pretreatment. Our results suggest that although CHOP is up-regulated by brefeldin A, it is not a major mediator of brefeldin A-induced apoptosis. collapse abstract
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DNA and cell biology 2006 Aug; 25(9)
PTX1(ERGIC2)-VP22 fusion protein upregulates interferon-beta in prostate cancer cell line PC-3.
PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It is unrelated to the pituitary homeobox protein (Ptx1 or Pitx1), which regulates pituitary hormone gene expre... expand abstractssion, and its function is currently unknown. Recently, it was found to be a homolog of the yeast Erv41p, an endoplasmic reticulum (ER) resident protein involved in protein trafficking between ER and Golgi, and was renamed as ERGIC2. Ectopic expression of a partial sequence of PTX1 (Met84 - Leu225) as a VP22-fusion protein in prostate cancer cell line, PC-3, induced cellular senescence. Gene expression microarray analyses showed that interferon-beta (IFN-beta) and a number of IFN-inducible genes, among other genes, were upregulated by the PTX1-VP22 fusion protein. Upregulation of IFN-beta was confirmed by RTPCR and promoter-reporter assay. However, the upregulation of IFN-beta by the PTX1-VP22 fusion protein was not due to nuclear translocation of the PTX1 luminal domain. collapse abstract
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DNA and cell biology 2003 Jun; 22(7)
Effects of PTX1 expression on growth and tumorigenicity of the prostate cancer cell line PC-3.
PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1... expand abstract cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells. collapse abstract
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DNA sequence : the journal of DNA sequencing and mapping 2001 Jun; 12(1)
Molecular cloning and sequence analysis of a human untranslated Alu-containing RNA.
A cDNA, designated LNX1, has been identified by subtractive hybridization on the basis that it is expressed in normal human prostate but not in LNCaP cells. Sequence analysis revealed that it contained two Alu repetitive sequences but no long open re... expand abstractading frame. Hence, it belongs to a class of untranslated Alu-containing RNAs. LNX1 is expressed in most tissues. It is encoded by a single copy gene, which is localized on human chromosome 14. collapse abstract
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DNA and cell biology 2001 May; 20(6)
Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer.
A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma. The full-length cDNA was subsequently established by 5' and 3' RACE. Nucleotide sequence analy... expand abstractsis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus. The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed. A segment of the cDNA was expressed in E. coli to produce a fragment of the PTX1 protein for the generation of specific antibodies. The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower. Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate. The nuclear staining was greatly reduced in prostate carcinoma. The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence. collapse abstract
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Molecular reproduction and development 2000 Nov; 57(4)
Expression and localization of prohormone convertase 13 (SPC3) in porcine ovary.
Tissue distribution and cellular localization of PC1/3 mRNA in porcine tissues were examined by ribonuclease protection assay and in situ hybridization. PC1/3 mRNA was detected mainly in the corpus luteum of pregnant sow and brain. Within the ovary, ... expand abstractPC1/3 and relaxin transcripts colocalized within large luteal cells. Levels of PC1/3 transcripts in corpora lutea increased as gestation advanced, parallel with an observed increase in relaxin transcripts. A role for PC1/3 in proprotein processing in the ovary is discussed. collapse abstract
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Journal of reproduction and fertility 1998 Aug; 114(1)
Immunostimulatory effects of pig seminal proteins on pig lymphocytes.
Pig seminal proteins PSP-I and PSP-II are major protein components of boars' ejaculate and are present as heterodimers (PSP-dimer) in seminal plasma. These proteins were examined for their ability to modulate pig lymphocyte activity in vitro in mitog... expand abstracten-induced lymphocyte proliferation assays and in one-way mixed lymphocyte reactions. Pig lymphocytes were cultured with phytohaemagglutinin, concanavalin A, or pokeweed mitogen (PWM) in the presence or absence of pig seminal proteins and the amount of cellular [3H]thymidine was used as an indication of proliferation. In the absence of mitogens, none of the three pig seminal proteins affected lymphocyte proliferation suggesting that these proteins are not antigenic or mitogenic. PSP-dimer enhanced lymphocyte proliferation induced by PWM (156-227%, P < 0.05) in a concentration-dependent manner, but had no effect on phytohaemagglutinin- or concanavalin A-induced proliferation. PSP-I enhanced (127-185%, P < 0.05) phytohaemagglutinin-induced proliferation. PSP-II augmented (130-240%, P < 0.05) lymphocyte proliferation induced by concanavalin A and PWM. Lymphocytes from gilts were significantly more responsive to concanavalin A- and PWM-induced lymphocyte proliferation in the presence of PSP-I compared with boars (concanavalin A: gilts 131%, boars 91%; PWM: gilts 188%, boars 134%; P < 0.05). In the mixed lymphocyte reaction, pretreating stimulating cells with increasing concentrations of PSP-I or PSP-II elicited a 400% concentration-dependent increase (P < 0.01) in lymphocyte proliferation. The abundance of pig seminal proteins in boar seminal plasma, their ability to enhance lymphocyte proliferation, and their previously reported ability to bind to lymphocytes suggest that these proteins are immunostimulatory and supports the hypothesis that they modulate uterine immune activity to ensure reproductive success. collapse abstract
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Biology of reproduction 1998 Jun; 59(1)
Purified porcine seminal plasma protein enhances in vitro immune activities of porcine peripheral lymphocytes.
The porcine seminal plasma protein (PSP) accounts for much more than 50% of the total proteins in seminal plasma. PSP has been previously purified and its biochemical properties characterized. However, the biological functions of PSP remain to be elu... expand abstractcidated. We hypothesize that PSP is involved in the regulation of uterine immune activity. In the current study, effects of PSP on in vitro lymphocyte activities and the presence of PSP binding sites on lymphocytes were examined. In mitogen-induced proliferation assay, lymphocytes from peripheral blood of gilts were cultured with pokeweed mitogen (PWM), phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of PSP. PSP at 50, 125, and 250 ng/well augmented PWM-induced [3H]thymidine uptake in a dose-responsive manner by 152.8 +/- 8.1%, 225.9 +/- 35.2%, and 274.8 +/- 53.6%, respectively, compared with that of control. PSP did not alter lymphocyte proliferation in the absence of PWM. Similarly, PSP had little or no effect on PHA- or Con A-induced lymphocyte proliferation. In one-way mixed lymphocyte reactions, PSP at 50, 125, and 250 ng/well enhanced [3H]thymidine uptake in a dose-responsive manner by 181.5 +/- 16.5%, 339.9 +/- 48.2%, and 600.1 +/- 84.8% of control, respectively. Using biotinylated PSP-I, PSP binding sites were localized on approximately 3-5% of the lymphocyte population. In summary, we have demonstrated that PSP itself is not a mitogen/antigen to porcine lymphocytes but that it has a stimulatory effect on lymphocyte activities initiated by PWM or surface antigens of lymphocytes. PSP may exert its functions by interacting with PSP binding sites on a subpopulation of porcine lymphocytes. The high potency of PSP on lymphocyte activities and the abundance of PSP in seminal plasma have suggested that PSP may play an important role in regulating immune responses in the porcine uterine environment. collapse abstract
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Endocrinology 1996 Oct; 137(11)
Prolactin-like protein-C variant: complementary deoxyribonucleic acid, unique six exon gene structure, and trophoblast cell-specific expression.
The rat placental PRL family consists of proteins structurally related to pituitary PRL. As a consequence of attempting to characterize the gene for one of the members of the family, PRL-like protein-C (PLP-C), we identified a related gene that we ha... expand abstractve termed PLP-C variant (PLP-Cv). In this study, we present information on the PLP-Cv gene and its pattern of expression. Screening of a rat genomic library with a PLP-C cDNA resulted in the isolation of four phage clones. Nucleotide sequence analysis of the clones revealed a gene, PLP-Cv, closely related but distinct from PLP-C. The PLP-Cv gene possessed a six exon/five intron organization, unique among members of the PRL family, and was localized to chromosome 17 of the rat genome, similar to other PRL family members. A PCR strategy involving primers based on the PLP-Cv gene was used to isolate a placental PLP-Cv cDNA. PLP-Cv showed 90 and 78% sequence identity with PLP-C at nucleotide and amino acid levels, respectively. Expression of PLP-Cv was restricted to the trophoblast lineage and was coordinately activated with PLP-C beginning at day 11 of gestation and continuing until term. Primer extension analysis revealed multiple putative transcription start sites. A 2.1-kilobase pair PLP-Cv promoter-luciferase reporter construct was specifically activated in differentiating rat trophoblast cells but not in other cell types. In conclusion, we have identified a new member of the PRL family possessing considerable homology to PLP-C, a unique gene structure, and displaying a trophoblast-specific pattern of transcriptional activation. collapse abstract
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Endocrinology 1996 Oct; 137(11)
Rcho-1 trophoblast cell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities.
In this report, we have investigated placental lactogens (placental lactogen-I, PL-I; PL-I variant, PL-Iv; PL-II) expressed by differentiated Rcho-1 trophoblast cells. A complementary DNA (cDNA) library to differentiated Rcho-1 trophoblast cells was ... expand abstractconstructed and screened with probes to detect PL-I and PL-II. Sequence analysis of three independent Rcho-1 PL-I cDNAs indicated that they significantly differed from the previously reported PL-I sequence but more closely resembled a related cDNA referred to as PL-I mosaic (PL-Im). Upon further analysis, Rcho-1 PL-I/PL-Im transcripts could be detected in Rcho-1 trophoblast cells and normal developing placental tissue; however, the previously reported PL-I transcript could not be identified from the same sources. Given these results, we examined the original PL-I cDNA by PCR and nucleotide sequence analyses. The sequence differed from the original report and was found to be identical to the Rcho-1 PL-I and PL-Im cDNA clones. Thus, PL-I, Rcho-1 PL-I, and PL-Im are equivalent and should be referred to as PL-I. The PL-I gene was localized to chromosome 17 of the rat genome, similar to other PRL family members. Rcho-1 PL-II cDNAs were identical to the published PL-II sequence. PL-Iv cDNAs were isolated from differentiated Rcho-1 cells via an RT-PCR strategy and found to be identical to previously isolated PL-Iv cDNAs. Rcho-1 PL-I and PL-II cDNAs were subcloned into the pcDNA3 expression vector and recombinant protein produced in HRP-1 cells. Both recombinant Rcho-1 PL-I and PL-II proteins significantly stimulated the proliferation of lactogen-dependent rat Nb2 lymphoma cells and mouse mammary epithelial cells. In summary, we show that the Rcho-1 PL-I corresponds to PL-Im and Rcho-1 PL-Iv and PL-II are identical to their previously described placental counterparts. Additionally, both recombinant Rcho-1 PL-I and PL-II proteins are biologically active. collapse abstract
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Molecular and cellular endocrinology 1996 Jan; 116(1)
Placental lactogen-I variant utilizes the prolactin receptor signaling pathway.
Placenta lactogen-I variant (PL-Iv) is a member of a family of proteins expressed by the rat placenta with characteristics similar to prolactin (PRL). In this report, we present the molecular cloning, chromosomal localization, and heterologous expres... expand abstractsion of PL-Iv. Nucleotide sequence analysis of the PL-Iv cDNA clone predicted a precursor protein of 223 amino acids, including a 28-amino acid signal sequence. The PL-Iv gene was localized to chromosome 17 of the rat genome, which also carries other members of the PRL gene family. PL-Iv heterologously expressed in Chinese Hamster ovary (CHO) cells exhibited similar immunoreactive and electrophoretic characteristics with PL-Iv produced by the rat placenta. N-terminal sequencing verified the identity and purity of the recombinant PL-Iv species and the site of cleavage of the signal peptide from the mature secreted PL-Iv species. Recombinant PL-Iv was shown to bind to ovarian and liver PRL receptors, stimulate the proliferation of Nb2 lymphoma cells, and activate Jak2. Each of these actions is consistent with PL-Iv utilizing the PRL receptor signal transduction pathway. collapse abstract
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Biochimica et biophysica acta 1995 Oct; 1264(1)
Characterization of multiple prohormone convertase PC13 transcripts in porcine ovary.
Overlapping cDNAs encoding porcine prohormone convertase, PC1/3, have been isolated from a pregnant sow ovary cDNA library using a mouse PC1/3 cDNA as a probe. Nucleotide sequence analysis of these cDNAs predicts a PC1/3 precursor protein of 753 amin... expand abstracto acid residues, which shares an overall sequence homology of 96, 92, and 92% with the human, rat, and mouse counterparts, respectively. Furthermore, five different polyadenylation sites have been observed. The utilization of these polyadenylation sites results in a length difference of 40-440 bp in the 3' untranslated regions of the transcripts. collapse abstract
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DNA and cell biology 1994 Mar; 13(4)
Molecular cloning and sequence analysis of the cDNA encoding porcine acrosin inhibitor.
A full-length cDNA encoding the porcine acrosin inhibitor has been isolated from a boar seminal vesicle cDNA library. Nucleotide sequence analysis of the 667-bp cDNA predicts a precursor protein of 97 amino acid residues, which includes a 26-residue ... expand abstractsignal peptide and a 71-residue secreted protein. The predicted amino acid sequence of the mature protein agrees completely with that of the sperm-associated acrosin inhibitor determined by conventional amino acid sequence analysis. However, the asparagine/aspartic acid and glutamine/glutamic acid substitutions, as reported in the seminal plasma counterpart, have not been observed. Southern blot analysis shows only a single hybridizing band with three different restriction endonucleases, suggesting the presence of a single copy of the acrosin inhibitor gene in the porcine genome. collapse abstract
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The Journal of biological chemistry 1994 Mar; 269(9)
Transcriptional activation of cytochrome P450 side chain cleavage enzyme expression during trophoblast cell differentiation.
The purpose of this study was to investigate P450scc expression during trophoblast differentiation. Biochemical characteristics of P450scc protein and mRNA identified in rat trophoblast tissues were similar to those identified in the rat adrenal glan... expand abstractd. Furthermore, P450scc was localized to trophoblast giant cells. This observation prompted an examination of progesterone biosynthesis and P450scc expression in Rcho-1 cells. Rcho-1 cells were derived from a transplantable rat choriocarcinoma, their differentiation can be regulated, and they have the capacity to express the trophoblast giant cell phenotype. Progesterone was produced by Rcho-1 cells and increased approximately 100-fold as the cells progressed from proliferation to differentiation. P450scc protein and mRNA accumulation also increased during trophoblast differentiation. P450scc expression within the Rcho-1 cell line was restricted to trophoblast giant cells. To further investigate the regulation of P450scc expression during trophoblast differentiation, we examined a plasmid construct, containing 894 base pairs of DNA 5' upstream from the P450scc transcriptional start site linked to a human growth hormone reporter gene, following stable transfection into Rcho-1 cells. The transfected P450scc regulatory DNA permitted the expression of human growth hormone which paralleled expression of the endogenous P450scc gene. In conclusion, transcriptional activation of the P450scc gene accompanies trophoblast giant cell differentiation. collapse abstract
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DNA and cell biology 1993 Aug; 12(7)
Molecular cloning and sequence analysis of two porcine seminal proteins, PSP-I and PSP-II: new members of the spermadhesin family.
Two full-length cDNAs encoding porcine seminal proteins, PSP-I and PSP-II, have been isolated from a porcine seminal vesicle cDNA library. Nucleotide sequence analysis of the 706-bp PSP-I cDNA predicts a precursor protein of 133 amino acid residues, ... expand abstractwhich includes a 21-residue signal peptide and a 112-residue secreted protein. On the other hand, the complete sequence of the 686-bp PSP-II cDNA reveals a coding sequence for a 21-residue signal peptide and a 116-residue secreted protein. The predicted amino acid sequences agree very well with those determined by conventional amino acid sequence analysis. Alignment of the two cDNA sequences shows an overall 66% sequence homology throughout their entire length. However, the sequence homology is much higher in the 3' untranslated region (72%) than in the coding region (61%). This suggests that these two genes evolved by duplication and divergence from a common ancestral gene. They share about 50% amino acid sequence homology and a similar overall structure with three members of the spermadhesin family. collapse abstract
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Molecular reproduction and development 1993 Jun; 35(3)
Binding characteristics and immunolocalization of porcine seminal protein, PSP-I.
PSP-I, a 13 kDa protein purified from boar seminal plasma, was found to have about 50% amino acid sequence homology with a family of zona pellucida-binding proteins known as spermadhesins. These proteins are produced by the accessory gland(s) of the ... expand abstractmale reproductive tract and coat the spermatozoa during ejaculation. In this study, we have investigated the possible biological functions of PSP-I using a solid-phase protein binding assay and its site of synthesis using both Western blot and immunocytochemical analyses. PSP-I was found to bind a number of proteins including endo-beta-galactosidase digested ZP3, soybean trypsin inhibitor, IgA, IgG and alpha-casein, indicating that it may have multiple functions. The protein or carbohydrate structures were not critical in the binding, since polyvinyl sulfate could effectively inhibit the binding of PSP-I to these proteins. Western blot analysis using specific antiserum to PSP-I showed that the protein was present in the seminal vesicle but not in the testes, epididymis or prostate. The protein was revealed by immunocytochemical analysis in the epithelium of seminal vesicles but not in the testes or the epididymis. It is concluded that PSP-I is synthesized by the epithelium of the seminal vesicles, secreted into the semen during ejaculation, and may be involved in various reproductive functions, such as preventing premature acrosome reaction and immunosuppression. collapse abstract
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Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 1993 Apr; 6(3)
Profile of prostatic-specific antigen in prostatic carcinomas.
Prostatic carcinomas (PCs) may be divided into two distinct categories: latent PCs, found mostly at autopsy, and clinical cases, which present with signs and symptoms. These two categories correspond fairly well to histologic grading of PCs and immun... expand abstractoperoxidase staining for prostatic specific antigen (PSA). The objective of this study was to find quantitative and qualitative differences if any, of PSA in PCs, corresponding to Gleason's histologic grade. By radiometric assay of PSA in tissue cytosol, PCs especially those of high histologic grade, were found to have lower PSA concentrations than normal and glandular hyperplastic prostatic tissue. Western blotting of cytosol was performed to detect differences between immunoreactive PSA of PCs compared with noncancerous tissue using both polyclonal and monoclonal antibodies against PSA. Western blotting with anti-PSA revealed some different bands between cancerous and noncancerous cytosols. Western blotting of cancerous and noncancerous cytosols was also performed using anti-prostatic acid phosphatase and anti-beta micro-seminoprotein. Reduced PSA concentration and different immunoblotting pattern of PSA were found to be characteristic for PCs, especially in carcinomas with grades higher than 7, which usually present with more aggressive invasion and metastases. collapse abstract
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Life sciences 52(12)
Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active.
Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studie... expand abstractd so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay. collapse abstract
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The Journal of biological chemistry 1993 Feb; 268(5)
Decidual prolactin-related protein. Identification, molecular cloning, and characterization.
In this report, we describe the isolation, molecular cloning, and characterization of a new member of the prolactin (PRL)-growth hormone (GH) family expressed in rat decidual tissue. A 29-kDa protein was isolated from medium conditioned by decidual e... expand abstractxplants. The protein possessed an affinity for concanavalin A and cross-reactivity with antibodies to two rat placental proteins, PRL-like protein-B (PLP-B) and PLP-C and with antibodies to human PRL. NH2-terminal sequencing of the isolated decidual protein indicated that it shared significant sequence identity with the NH2 terminus of PLP-C. The decidual protein was termed decidual prolactin-related protein (dPRP). A PLP-C cDNA was used to identify dPRP cDNAs from a rat decidual cDNA library. Nucleotide sequence analyses of the dPRP cDNAs predicted a mature protein of 239 amino acids, including a 28-amino acid signal sequence. The predicted dPRP amino acid sequence contains two putative N-linked glycosylation sites and 6 cysteine residues. The 6 cysteines are located in positions homologous to the cysteines of PLP-C and PRL. Additional sequence similarities with members of the PRL-GH family are evident. The dPRP gene was localized to rat chromosome 17, which also carries other members of the PRL gene family. Northern blot analysis showed that the dPRP cDNA clone specifically hybridized to a 1.0-kilobase mRNA. The relationship of dPRP with other members of the PRL-GH family and its putative role(s) in the physiology of pregnancy are discussed. collapse abstract
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The Journal of biological chemistry 1993 Feb; 268(5)
Heterologous expression and characterization of prolactin-like protein-A. Identification of serum binding proteins.
In this report, we describe the heterologous expression of prolactin-like protein-A (PLP-A) in Chinese hamster ovary (CHO) cells, the characterization of recombinant PLP-A, and the identification of serum PLP-A-binding proteins. CHO cell and native p... expand abstractlacental PLP-A showed similar immunoreactive characteristics and electrophoretic mobilities. N-terminal sequencing verified the identity and purity of the recombinant PLP-A species and the site of cleavage of the signal peptide from the mature secreted PLP-A species. Recombinant PLP-A lacked activity in standardized prolactin and growth hormone in vitro bioassays. Antibodies generated to recombinant PLP-A facilitated the cellular localization of PLP-A and the identification of high molecular weight PLP-A complexes. Cross-linking analyses of radioiodinated PLP-A with serum harvested from late gestation rats indicated the presence of two major cross-linked complexes migrating under reducing conditions at 130 and 250 kDa and two minor cross-linked complexes migrating at 70 and 110 kDa. Binding of PLP-A to serum proteins was specific for PLP-A and not effectively competed by other members of the prolactin/growth hormone family. The PLP-A binding species were also found in serum from non-pregnant female and male rats. collapse abstract
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Archives of biochemistry and biophysics 1992 May; 295(2)
Purification and characterization of PSP-I and PSP-II, two major proteins from porcine seminal plasma.
Two major glycoproteins, designated PSP-I and PSP-II, were purified from porcine seminal plasma by ammonium sulfate fractionation, CM-cellulose chromatography, gel filtration on Sephadex G-75 (superfine), and reverse phase high performance liquid chr... expand abstractomatography. These two proteins exist in several forms differing mainly in the carbohydrate moiety. The complete amino acid sequence of PSP-I has been determined by automated Edman degradation of peptides generated by proteolytic digestion and cyanogen bromide cleavage. The protein is 109 residues long and has a single glycosylation site at the asparagine residue at position 47. In addition, the N-terminal sequence of PSP-II has also been determined. PSP-I is a unique protein; a sequence homology search using the protein data base did not reveal any significant homology with other proteins. PSP-II shares 50% sequence homology with a family of zona pellucida-binding glycoproteins at the N-terminus. collapse abstract
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Archives of biochemistry and biophysics 1992 Apr; 294(2)
Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.
In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinan... expand abstractt plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin. collapse abstract
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