Pancreatic spasmolytic polypeptide (PSP): I. Preparation and initial chemical characterization of a new polypeptide from porcine pancreas

A novel polypeptide, named Pancreatic Spasmolytic Polypeptide (PSP), was discovered in a side-fraction from the purification of porcine insulin. PSP was prepared by two different purification methods based on combinations of precipitations, anion-exchange and cation-exchange chromatography. The highest yield obtained, 52 mg PSP/kg pancreas, indicates that the content of PSP in porcine pancreas is about half the content of insulin. Both preparations appeared to be very pure as judged by basic disc electrophoresis, isoelectric focusing, analytical gel filtration and radioimmunoassays for various polypeptides known to be present in pancreas. The PSP molecule contains 106 amino acids (MW about 11 700). PSP is an acidic (pI 4.4), non-glycosylated protein without free N-terminal amino groups, and with high contents of proline and cystine. The high content of S-S bridges (7 per molecule), an unexpected low apparent MW determined by gel filtration, and a remarkable resistance towards treatment with trypsin and chymotrypsin, point to a compact structure of the PSP molecule.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig

Summary Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Purification,characterization and developmental expression of pig liver PSP

We have isolated a perchloric acid-soluble protein designated as PL-PSP from the post-mitochondria supernatant fraction of pig liver. It is soluble in 5% perchloric acid and purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The PL-PSP showed approximately 80–90% homology with PSP isolated from rat liver (RL-PSP) with its partial amino acid sequences. The protein has a molecular mass of approximately 14 kDa which was slightly higher than that of RL-PSP. It inhibited protein synthesis in a rabbit reticulocyte lysate system. The expression of PL-PSP was predominant in liver, kidney and duodenum, and was also expressed in stomach, lung and brain. PL-PSP expression in liver increased from the 1st day to the 1st month. Thus, our findings are the first report on the presence of a PSP in porcine tissues which may be involved in the regulation of cellular growth and differentiation.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig.

Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Recombinant PSP94 (prostate secretory protein of 94 amino acids) demonstrates similar linear epitope structure as natural PSP94 protein

PSP94 has the potential to be a useful diagnostic marker and therapeutic agent in prostate cancer. Recently, different immunoassay systems for quantitative analysis of PSP94 in clinical samples have been developed, but the epitope structure of PSP94 protein has not been elucidated. In this study, we report an Escherichia coli expression system for recombinant GST-PSP94 fusion protein. GST-PSP94 contains antigenic determinants similar to natural PSP94 protein (determined both by Western blotting experiments and by ELISA) and can be used to study the structure of natural PSP94 antigen. Since GST-PSP94 was expressed in E. coli and purification involved a denaturing process, we propose that the epitope structure of PSP94 is linear and largely dependent on the primary amino acid sequence, rather than conformational structure. This hypothesis was supported by reciprocal competition in ELISA among natural, GST-PSP94 fusion protein, and purified recombinant PSP94 protein. The results demonstrate that the various forms of PSP94 can compete with each other in binding to rabbit PSP94 polyclonal antibody, although the natural PSP94 has a slightly higher affinity. When natural and recombinant PSP94 protein were denatured in vitro with urea and alkali, no effect on the binding to antibody was found. The epitope activity of natural PSP94 was also shown to be resistant to the treatment of detergent and reducing agent. The location of one of the linear epitopes recognized by the PSP94 antibody was determined to be in the N-terminus by using two synthetic peptides representing N- and C-terminal sequences. Competitive ELISA between the N-terminal peptide and PSP94 protein indicate that both natural and GST-PSP94 have similar immunoactive N-termini. © 1996 Wiley-Liss, Inc.     

人PSP94的分泌表达及其表达产物抗前列腺癌作用

将编码人 94个氨基酸的前列腺分泌蛋白 ( PSP94) c DNA与酵母整合载体 p PICZαA重组 ,构建的重组质粒线性化后转染酵母细胞 GS1 1 5,获得了 PSP94在酵母细胞中遗传性稳定表达酵母工程细胞 .诱导后的培养物中 ,rh PSP94表达量约为 0 .9mg/L,分子量约 1 6.5k D.培养上清经离子交换层析纯化后 ,目的蛋白的纯度为 92 % .体外在人前列腺癌细胞上活性分析表明 ,rh PSP94以1 0 0μg/L ,对该细胞的抑制率 2 0 .4% ;单纯新型 TNF,以 1 0 3 U/ml,抑制率 2 9.8% ;rh PSP94和新型 TNF以上述同样剂量联合应用 ,抑制率为 86.3% .提示 PSP94在体外对抗前列腺癌细胞有杀伤作用 ,但不明显 ;PSP94与新型 TNF联合应用 ,可使抑制率明显提高 ,可能 PSP94与新型 TNF有协同抗前列腺癌的作用 .     

Interannual variability of PSP outbreaks on the north east UK coast

Paralytic shellfish poisoning (PSP) occurs sporadically on theNB UK coast. The degree of toxicity shows considerable interannualvariability, but particularly severe events occurred in 1968and 1990. The time sequence of PSP toxin production in 1990is described and compared with 1989 when no significant PSPtoxin occurred. In 1990, PSP toxin was widespread in shellfishsamples taken on 300 km of coastline, from Berwick to Whitby,and toxin was present at high concentrations for >1 month.The distribution of Alerandrium tamarense cysts in the sedimentsis described. High concentrations were found in the Firth ofForth and also in a number of regions offshore of the Scottishand English coasts. A water transport model has been used toestimate back trajectories, with the aim of determining thesource of the A.tamarense bloom. The Firth of Forth has previouslybeen suggested as the seed bed for A.tamarense outbreaks inthe area, but the transport model clearly shows that A.tanwrensemoved inshore over a wide area in 1990; there was no singlesource of the bloom. Sea surface temperatures, estimated fromsatellite imagery, show that water temperatures were much higherat the end of April 1990, when the bloom occurred, than in 1989when PSP toxin incidence was very low. These conditions wouldhave resulted in early seasonal stratification and would havefavoured phytoplankton growth in the water column.     

Paralytic shellfish poisoning (PSP) in Margarita Island, Venezuela

A severe outbreak of Paralytic Shellfish Poisoning (PSP) occurred in Manzanillo and Guayacán, northwestern coast of Margarita Island, Venezuela, between August and October 1991. A bloom of dinoflagellates including Prorocentrum gracile, Gymnodinium catenatum and Alexandrium tamarense seemed to be responsible for this outbreak. Levels of PSP toxins in mussels (Perna perna) exceeded the international safety limit of saxitoxin, 80 microg STX/100 microg meat. PSP toxin values varied between 2548 and 115 microg STX/100 g meat in Manzanillo, and between 1422 and 86 microg STX/100 g meat in Guayacán. At both locations, the highest levels were detected in August, when 24 patients exhibited typical symptoms of PSP toxicity after consuming cooked mussels (16 required hospitalization). A high pressure liquid chromatographic (HPLC) procedure was recently used on the 1991 samples. The major toxin detected in samples of both locations was decarbamoyl saxitoxin (dcSTX), but low concentrations of saxitoxin were also found in Manzanillo samples. Gonyautoxins GTX1, GTX2 and GTX3 were detected only at Guayacán, while in both locations, decarbamoylgonyatouxin (dcGTX2,3) toxins were detected. These findings represent the first time that causative toxins of PSP in Venezuela have been chemically identified, and confirm the presence of dcSTX and dcGTX in mussels from the Caribbean Sea. The presence of dcSTX and dcGTX in shellfish is indicative that Gymnodinium catenatum was a causative organism for outbreak of PSP.     

A New Scalable Purification Procedure for Prostatic Secretory Protein (PSP94) from Human Seminal Plasma

A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS–polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18–25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS–PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS–PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.     

Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin

Prostate secretory protein of 94 amino acids (PSP94) is a small non-glycosylated, cysteine rich protein with a molecular mass of 10 kDa. It has also been referred to as beta-microseminoprotein (beta-MSP) and proteins homologous to it have been reported in a number of species. Comparison of the amino acid sequence of these proteins suggests that, it is a rapidly evolving protein. However, all the ten cysteine residues are well conserved in these homologues, indicating their possible role in maintaining the structure and function of these proteins. In the present study, PSP94 was purified from human seminal plasma and characterized further and it showed the presence of five disulfide bonds. Reduction of disulphide bonds of PSP94 led to significant changes in the secondary and tertiary structure of PSP94. CD of disulphide bond reduced PSP94 indicates an overall decrease in the beta sheet content from 79.8% to 46.4%. Tertiary structural changes as monitored by fluorescence quenching reveal that reduction of disulphide bonds of PSP94 followed by the modification of the free thiol groups leads to complete exposure of Trp32 and Trp92 and that one or more side chain carboxyl groups move closer to their indole side chains. Antibodies against native and modified PSP94 demonstrated that the changes following reduction of disulphide linkages are within the immunodominant region of the protein. Changes induced in the functional properties of PSP94, if any, by modification were investigated with respect to IgG binding as PSP94 has been reported to be similar to immunoglobulin binding factor purified from seminal plasma. A novel finding from this study is that both native PSP94 as well as modified protein have the ability to bind human IgG, suggesting the involvement of sequential epitopes of PSP94 in IgG binding.     

人重组PSP及其生物活性研究

为了获得人重组 persephin( PSP)并研究其生物学活性 ,从人胎脑组织中提取总 RNA,以RT- PCR方法获取编码人 PSP成熟蛋白 c DNA.将人 PSP c DNA插入含 T7启动子的质粒 p ET-2 8a( + ) ,构建表达质粒 p ET- PSP,转化大肠杆菌 BL 2 1 ( DE3)获得表达菌株 BLPSP,经 IPTG诱导表达的 PSP形成包含体 .凝胶自动扫描分析表明 ,PSP表达量约占菌体总蛋白 2 0 %以上 .用Ni2 + - NTA树脂一步法纯化目的蛋白 ,纯度达 85%以上 .纯化和复性的 PSP蛋白能显著促进脊髓神经元的存活 .     

First report of paralytic shellfish poisoning (PSP) caused by Alexandrium tamiyavanichii in Kuantan Port,Pahang, East Coast of Malaysia

Harmful algal bloom (HAB) is a proliferation of algae, which naturally produce biotoxins and cause harmful effects to humans, the environment and organisms associated with it. Paralytic shellfish poisoning (PSP) was reported for the first time in Kuantan Port, Pahang, Malaysia, in November 2013, followed by a second episode in August 2014. The toxicity level reported during the second event was as high as 3500 μg of STX equiv./100 g shellfish. Ten people were hospitalized with PSP symptoms after consuming contaminated shellfish. This study was conducted at Kuantan Port to identify the organisms responsible for these events. Water samples were collected monthly for a period of 12 months beginning in September 2014. HAB species were identified based on their morphology using light and fluorescence microscopes, and their classification was supported by molecular evidence based on internal transcribed spacer (ITS) sequences. Monthly cell abundance of Alexandrium tamiyavanichii was measured at four sampling stations. Toxin production by three strains isolated from the area was determined using HPLC. Our results revealed the presence of several HAB species, including the PSP‐producing species A . tamiyavanichii . The highest cell density of A . tamiyavanichii was 840 cells L^?1. The presence of GTX components was detected in these strains. However, other toxin components could not be determined. This study reported, for the first time, the presence of PSP‐producing A . tamiyavanichii on the Pahang coast of east Peninsular Malaysia and confirmed that the PSP events in Kuantan Port were attributable to this species. The presence of this species further indicates that several safety measures need to be considered to safeguard public health, particularly in Pahang coastal waters.     

Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients

PSP94 (prostate secretory protein of 94 amino acids) was regarded as a possible prostate cancer marker, however, it has been controversial. All prior studies were designed to test the free form in serum using antibodies to PSP94. Results presented here demonstrate that PSP94 exists in prostate cancer patients in two forms, free and bound, and that the majority is present as serum bound complexes. This result was demonstrated by using both native and SDS-PAGE analyses of serum proteins from prostate cancer patients. Chromatographic separation of serum total proteins by a molecular sieve column generated two peaks (peak I and II), which were reactive with rabbit antiserum to human PSP94 in Western blot experiments. Peak I was eluted before the IgG fraction at a molecular weight larger than 150 kDa, and peak II appeared after serum albumin ( approximately 67 kDa) was eluted. By using a biotinylated PSP94 as an indicator of the free form of PSP94, we demonstrate that peak I contains serum PSP94-bound complexes and peak II is likely the free form of serum PSP94. Since the molecular weight of serum PSP94-bound complexes is close to IgG during molecular sieve separation, serum PSP94 complexes were further purified through two rounds of protein A column separation, followed by DEAE-ion exchange column chromatography. In vitro dissociation tests of the purified PSP94-bound complexes showed that the binding of serum PSP94-complexes is probably via disulfide bonds and is chemically stable. The results presented here indicate that serum PSP94-bound complexes must be considered in evaluating the clinical utility of PSP94 as a prostate cancer marker.     

Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients

PSP94 (prostate secretory protein of 94 amino acids) was regarded as a possible prostate cancer marker, however, it has been controversial. All prior studies were designed to test the free form in serum using antibodies to PSP94. Results presented here demonstrate that PSP94 exists in prostate cancer patients in two forms, free and bound, and that the majority is present as serum bound complexes. This result was demonstrated by using both native and SDS‐PAGE analyses of serum proteins from prostate cancer patients. Chromatographic separation of serum total proteins by a molecular sieve column generated two peaks (peak I and II), which were reactive with rabbit antiserum to human PSP94 in Western blot experiments. Peak I was eluted before the IgG fraction at a molecular weight larger than 150 kDa, and peak II appeared after serum albumin (∼67 kDa) was eluted. By using a biotinylated PSP94 as an indicator of the free form of PSP94, we demonstrate that peak I contains serum PSP94‐bound complexes and peak II is likely the free form of serum PSP94. Since the molecular weight of serum PSP94‐bound complexes is close to IgG during molecular sieve separation, serum PSP94 complexes were further purified through two rounds of protein A column separation, followed by DEAE‐ion exchange column chromatography. In vitro dissociation tests of the purified PSP94‐bound complexes showed that the binding of serum PSP94‐complexes is probably via disulfide bonds and is chemically stable. The results presented here indicate that serum PSP94‐bound complexes must be considered in evaluating the clinical utility of PSP94 as a prostate cancer marker. J. Cell. Biochem. 76:71–83, 1999. © 1999 Wiley‐Liss, Inc.     

Chemopreventive effect of PSP through targeting of prostate cancer stem cell-like population

Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.     

Accumulation of PSP toxins in Atlantic mackerel: seasonal and ontogenetic variations

Atlantic mackerel Scomber scombrus are known to be lethal vectors of paralytic shellfish poisoning (PSP) toxins to predators. To elucidate dynamics of PSP toxin accumulation in this species, mackerel were sampled in the Gulf of St Lawrence from May to October 1993. Mackerel appear to retain toxins (saxitoxin, gonyautoxins 2 and 3) year-round. The toxin content of the liver, as determined by high performance liquid chromatography, increased significantly with fish age ( r² =0.40) and length ( r² =0.52), suggesting that mackerel progressively accumulate PSP toxins throughout their life. The toxin content of the liver also increased significantly during the summer feeding sojourn in the Gulf of St Lawrence. Comparison of profiles of saxitoxin derivatives indicated that zooplankton were the likely source of PSP toxins found in mackerel. The mean ± S.D toxin content was 17.4 ± 10.6 nmol liver⁻¹ and the mean ± S.D. PSP toxicity was 112.4 ± 67.0 μg saxitoxin equivalents 100 g⁻¹ liver wet weight ( n =247).     

PSP94-TNFαD11a融合基因在NIH3T3细胞中的表达及其产物活性分析

 为了分析 PSP94- TNFαD1 1 a融合基因的表达和表达产物的生物学活性 ,将含该融合基因的质粒 pc DNA- PSP94- TNFα D1 1 a转染 NIH3T3细胞 ,72 h后收集细胞培养上清 ,并提取细胞总RNA,经 RT- PCR,得到与目的基因长度相符合的 c DNA片段 ;以 PSP94c DNA为探针 ,对 RT-PCR产物进行 Southern印迹分析 .结果表明 :转染 PSP94- TNFαD1 1 a融合基因的 NIH3T3细胞 ,其 RT- PCR产物杂交信号为阳性 .细胞培养上清用 TNF抗体行 Western印迹和 ELISA分析 ,检测结果为阳性 .生物学活性分析表明 ,细胞培养上清不仅具有 PSP94抑制人前列腺癌细胞 PC- 3生长的活性 ,而且显示出 TNFα对 L92 9细胞的细胞毒作用 .以上结果表明 ,pc DNA- PSP94- TNFαD1 1 a质粒能够正确表达目的基因 PSP94- TNFα D1 1 a,且表达的 PSP94- TNFαD1 1 a融合蛋白具有预期的双重生物学活性 .     

Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94.

Prostate secretory protein (PSP94, 94 amino acids) is one of the most abundant proteins secreted from the prostate. Its biological role is unknown and still controversial, although it is assumed to have the potential to be a biomarker and a suppressor of prostate cancer. In order to establish an animal model to further elucidate its biological role, we expressed the mature form of rat PSP94 in Escherichia coli, using a glutathione S-transferase (GST) fusion expression vector; we generated a polyclonal rabbit antibody against the recombinant protein. The antibody specifically recognized recombinant rat PSP94 and cross-reacted only very weakly with its human homologue. Using the characterized anti-rat PSP94 antibody, we found that PSP94 was located primarily in rat prostate. Furthermore, PSP94 is present at different levels in different lobes of rat prostate, with significant levels detectable only in the lateral lobe (LP). In addition, the most abundant PSP94 expression was found in the prostate lobe secretions, and PSP94 levels in LP secretions were at least seven times higher than in secretions from the dorsal prostate (DP). The rat ventral prostate (VP) and other regions of the male accessory glands were found to be almost completely devoid of PSP94. Since most rat prostate dysplasia induced by steroid hormone treatment occurs only in dorsolateral prostate, prostate tissue-specific expression and the expression of PSP94 in dorsolateral, but not other, lobes of the prostate suggest a potential role in prostate targeting and prostate cancer development.     

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.