Human sperm cells can form paracetamol metabolite AM404 that directly interferes with sperm calcium signalling and function through a CatSper-dependent mechanism.

STUDY QUESTION: Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? SUMMARY ANSWER: While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. WHAT IS KNOWN ALREADY: Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). LIMITATIONS, REASONS FOR CAUTION: The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. WIDER IMPLICATIONS OF THE FINDINGS: We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. STUDY FUNDING/COMPETING INTEREST(S): D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

The prognostic importance of miR-21 in stage II colon cancer: a population-based study.

BACKGROUND: Despite several years of research and attempts to develop prognostic models a considerable fraction of stage II colon cancer patients will experience relapse within few years from their operation. The aim of the present study was to investigate the prognostic importance of miRNA-21 (miR-21), quantified by in situ hybridisation, in a unique, large population-based cohort. PATIENTS AND METHODS: The study included 764 patients diagnosed with stage II colon cancer in Denmark in the year 2003. One section from a representative paraffin-embedded tumour tissue specimen from each patient was processed for analysis of miR-21 and quantitatively assessed by image analysis. RESULTS: The miR-21 signal was predominantly observed in fibroblast-like cells located in the stromal compartment of the tumours. We found that patients expressing high levels of miR-21 had significantly inferior recurrence-free cancer-specific survival (RF-CSS): HR=1.26; 95% CI: 1.15-1.60; P<0.001. In Cox regression analysis, a high level of miR-21 retained its prognostic importance and was found to be significantly related to poor RF-CSS: HR=1.41; 95% CI: 1.19-1.67; P<0.001. CONCLUSION: The present study showed that increasing miR-21 expression levels were significantly correlated to decreasing RF-CSS. Further investigations of the clinical importance of miR-21 in the selection of high-risk stage II colon cancer patients are merited.

Systemic administration of anti-urokinase plasminogen activator receptor monoclonal antibodies induces hepatic fibrin deposition in tissue-type plasminogen activator deficient mice.

BACKGROUND: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and hence pro-u-PA activation, is an attractive approach to anti-invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u-PAR (mR1) by immunization of u-PAR-deficient mice. OBJECTIVES: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. METHODS: Wild-type and tissue-type plasminogen activator (t-PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. RESULTS: Systemic administration of mR1 caused significantly increased fibrin signal in anti-u-PAR treated t-PA-deficient mice compared to mock-treated, which mimics the phenotype of u-PAR;t-PA double-deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t-PA-deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u-PAR. CONCLUSION: We show that u-PAR-expressing macrophages are involved in cell-mediated fibrinolysis of liver fibrin deposits, and that the antimouse-u-PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u-PA/u-PAR interaction in mouse cancer models.

Direct evidence of the importance of stromal urokinase plasminogen activator (uPA) in the growth of an experimental human breast cancer using a combined uPA gene-disrupted and immunodeficient xenograft model.

Several studies have indicated an interaction between tumor cells and infiltrating stromal cells regarding the urokinase plasminogen activation (uPA) system. By developing combined uPA gene-disrupted and immunodeficient mice, we have studied the role of stromal uPA for the growth of the MDA-MB-435 BAG human tumor xenograft. Subcutaneous tumor growth and lung metastasis were compared between wild-type immunodeficient mice and mice with the combined deficiencies. Tumor growth was evaluated by volume measurements and plasma beta-galactosidase activity and metastasis was evaluated by counting lung surface metastases. Although no differences appeared in primary tumor take between the two groups of mice, a significant difference was observed in primary tumor growth, with tumors in uPA-/- mice growing significantly more slowly. In addition, a nonsignificant trend toward fewer lung metastases in uPA-/- mice was observed. The present data points to a critical role of stromal-derived uPA in the primary tumor growth of MDA-MB-435 BAG xenografts, whereas only a trend toward fewer lung metastases in uPA gene-disrupted mice was found.

Collagenase-3 expression in breast myofibroblasts as a molecular marker of transition of ductal carcinoma in situ lesions to invasive ductal carcinomas.

Collagenase-3 (matrix metalloproteinase 13; MMP-13), a protease originally identified in breast carcinoma, is characterized by a potent degrading activity against a wide spectrum of extracellular matrix proteins. The aims of this study were to localize and identify the MMP-13-expressing cells in invasive human breast carcinoma and to evaluate the role of MMP-13 in transition to invasive lesions by studying ductal carcinoma in situ (DCIS). We found expression of MMP-13 in stromal fibroblast-like cells in all 21 invasive ductal carcinomas studied and in 4 of 9 invasive lobular carcinomas. In most carcinomas, expression of MMP-13 was limited to small stromal foci in the tumor area. Combined in situ hybridization and immunohistochemistry showed coexpression of alpha-smooth muscle actin immunoreactivity and MMP-13 mRNA in myofibroblasts. In contrast, cytokeratin-positive cancer cells, alpha-smooth muscle actin-positive vascular smooth muscle cells, CD68-positive macrophages, and CD31-positive endothelial cells were all MMP-13 mRNA negative. In situ hybridization for MMP-13 in 17 DCIS lesions revealed expression in 10 cases. Immunohistochemical analysis of all DCIS cases identified microinvasion in 8 of the 17 lesions. Seven of the eight lesions with microinvasion were MMP-13 positive. Further analysis showed that MMP-13 expression was often associated with the microinvasive events. This particular expression pattern was unique for MMP-13 among other MMPs analyzed, including MMP-2, -11, and -14. We conclude that MMP-13 is primarily expressed by myofibroblasts in human breast carcinoma and that expression in DCIS lesions often is associated with microinvasive events. On the basis of these data, we propose that MMP-13 may play an essential role during transition of DCIS lesions to invasive ductal carcinomas.

Sequential coexpression of the multidrug resistance genes MRP and mdr1 and their products in VP-16 (etoposide)-selected H69 small cell lung cancer cells.

Resistance to drugs included in the multidrug-resistance phenotype has been attributed to overexpression of either mdr1 or MRP genes and their products in numerous cell lines, while coexpression, to our knowledge, has not previously been reported in the same cells. Human small cell lung cancer H69/VP cells were developed by continuous incubation in increasing doses of VP-16. In reverse transcription-PCR assays we found over-expression of both mdr1 and multidrug-resistance protein (MRP) genes, and immunoblots showed both elevated P-glycoprotein and MRP in H69/VP cells. Double immunocytochemical staining demonstrated the expression of both MRP and P-glycoprotein in the same cells, indicating that the observations do not result from the selection of two independent clones. Examination of early passages of H69/VP cells showed that overexpression of MRP mRNA occurred prior to mdr1. Thus, cell lines and clinical samples in the future should be tested for both mdr1/P-glycoprotein and MRP since a positive result for one of the phenotypes does not preclude the existence of the other.

Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice.

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen-deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.

The urokinase receptor associated protein (uPARAP/endo180): a novel internalization receptor connected to the plasminogen activation system.

The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While the biological consequences of this reaction have not yet been verified experimentally, a likely event is ligand internalization because uPARAP is a constitutively recycling internalization receptor. uPARAP also binds at least one component, collagen type V, in the extracellular matrix meshwork, pointing to a potential role in proteolytic substrate presentation. Additional ligands have been proposed, including collagenase-3 and glycoproteins capable of interacting with one of the multiple carbohydrate recognition-type domains of uPARAP. In various adult tissues uPARAP is present on fibroblasts, macrophages and a subset of endothelial cells. In fetal tissues the protein has also been demonstrated in certain bone forming regions. Hypotheses on the physiological function of uPARAP include regulatory roles in extracellular proteolysis. This type of function would be likely to direct the local turnover of proteases and their substrate degradation products and thus may add to the complicated interplay between several cell types in governing restricted tissue degradation.

DUF1220 protein domains drive proliferation in human neural stem cells and are associated with increased cortical volume in anthropoid primates.

Genome sequences encoding DUF1220 protein domains show a burst in copy number among anthropoid species and especially humans, where they have undergone the greatest human lineage-specific copy number expansion of any protein coding sequence in the genome. While DUF1220 copy number shows a dosage-related association with brain size in both normal populations and in 1q21.1-associated microcephaly and macrocephaly, a function for these domains has not yet been described. Here we provide multiple lines of evidence supporting the view that DUF1220 domains function as drivers of neural stem cell proliferation among anthropoid species including humans. First, we show that brain MRI data from 131 individuals across 7 anthropoid species shows a strong correlation between DUF1220 copy number and multiple brain size-related measures. Using in situ hybridization analyses of human fetal brain, we also show that DUF1220 domains are expressed in the ventricular zone and primarily during human cortical neurogenesis, and are therefore expressed at the right time and place to be affecting cortical brain development. Finally, we demonstrate that in vitro expression of DUF1220 sequences in neural stem cells strongly promotes proliferation. Taken together, these data provide the strongest evidence so far reported implicating DUF1220 dosage in anthropoid and human brain expansion through mechanisms involving increasing neural stem cell proliferation.

An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator.

Using 3' RACE (rapid amplification of cDNA ends), we have isolated a cDNA variant for the receptor for human urokinase plasminogen activator (uPAR). The deduced protein includes the amino-terminal ligand binding domain in uPAR, but lacks the carboxy-terminal membrane attachment by a glycolipid anchor. Genomic DNA analysis showed that the uPAR mRNA variant is generated by alternative splicing. The new variant mRNA is expressed in various human cell lines and tissues and both variants are up-regulated by phorbol ester in A549 cells. We propose that the alternatively spliced uPAR mRNA encodes a soluble uPA binding protein, the possible function of which is discussed.

Quality control of immunohistochemical evaluation of tumour-associated plasminogen activators and related components. European BIOMED-1 Concerted Action on Clinical Relevance of Proteases in Tumour Invasion and Metastasis.

The plasminogen activation (PA) system is involved in the breakdown and remodelling of the extracellular matrix. In the case of cancer, this is a prerequisite for invasion and metastasis. The expression of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 in particular have been reported to be of clinical and prognostic value. This has primarily been proven in the case of breast carcinoma and colon carcinoma, using the enzyme-linked immunosorbent assay (ELISA) as a quantitative assay to determine the level of expression. Immunohistochemistry is another technique to investigate the presence of PA components. It allows assessment in a semiquantitative way and informs in addition on the specific distribution within the tissue. To take full advantage of the benefits of immunohistochemistry, it is important to aim at optimal quality in all steps influencing the final judgement of the staining results. These various steps are highlighted and discussed in this paper.

The murine receptor for urokinase-type plasminogen activator is primarily expressed in tissues actively undergoing remodeling.

uPAR is a cellular receptor for urokinase plasminogen activator, an enzyme involved in extracellular matrix degradation during processes involving tissue remodeling. We have expressed a recombinant soluble form of murine uPAR and raised rabbit polyclonal antibodies to study the expression of uPAR by immunohistochemistry. The immunohistochemical localization of uPAR was determined in normal mouse organs and in tumors formed by the highly metastatic Lewis lung carcinoma. uPAR immunoreactivity was found in the lungs, kidneys, and spleen, and in endothelial cells in the uterus, urinary bladder, thymus, heart, liver, and testis. No uPAR immunoreactivity was detected in muscle. In general, strong uPAR immunoreactivity was observed in organs undergoing extensive tissue remodeling, as exemplified by trophoblast cells in placenta, and in migrating, but not resting, keratinocytes at the edge of incisional wounds. Staining was not detected in any tissue sections derived from uPAR-deficient mice, thus confirming the specificity of the immunohistochemical staining of uPAR in normal mouse tissues. In Lewis lung carcinoma, uPAR immunoreactivity was found in the tumor cells of the primary tumor and in lung metastases. (J Histochem Cytochem 49:237-246, 2001)

Immunohistochemical identification of the receptor for urokinase plasminogen activator associated with fibrin deposition in normal and ectopic human placenta.

The receptor for urokinase plasminogen activator (uPAR) is a key molecule in cell surface-directed plasminogen activation. uPAR binds urokinase plasminogen activator (uPA) and thereby focuses plasminogen activation on the cell surface. Plasmin dissolves fibrin deposits and facilitates cell migration during tissue repair processes by degrading the extracellular matrix. During human implantation and placental development, plasmin is considered important for both trophoblast migration/invasion and for fibrin surveillance. This study examined the expression of uPAR in normal and ectopic human placentae by immunohistochemistry. In first and third trimester normal placentae as well as in tubal ectopic placental tissues, a high uPAR expression was seen in the trophoblast associated with deposits of fibrin-type fibrinoid. Extravillous trophoblast of the basal plate, of the cell islands, and of the cell columns was also positive for uPAR in the first trimester whereas at term the expression of the protein was decreased. Moreover, uPAR immunostaining was observed in decidual cells throughout normal gestation and in endometrial tissues of patients with ectopic pregnancies. These findings suggest that uPAR participates in placental development and in trophoblast invasion particularly in the first trimester of pregnancy and that uPAR is involved in repair mechanisms of the trophoblast and fibrin surveillance.

Cancer invasion and tissue remodeling--cooperation of protease systems and cell types.

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non-neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI-1 and MMPs) can be expressed by either the non-neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non-neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors.

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.

Cholesterol-lowering diets may increase the food costs for Danish children. A cross-sectional study of food costs for Danish children with and without familial hypercholesterolaemia.

Food costs for 30 children under dietary treatment for familial hypercholesterolaemia were compared with those of 105 other Danish children. The daily intake of macronutrients and the daily cost of the diet for each child were calculated from dietary intakes and average prices of 365 different food items. The mean +/- SE percentages of energy (E%) from fat in the diet of children with and without known familial hypercholesterolaemia were 23.6 +/- 0.8 E+ and 34.5 +/- 0.5 E%, respectively (P < 0.001). The dietary costs per MJ in these two groups were 3.79 +/- 0.12 Danish crowns (DKr) and 3.34 +/- 0.05 DKr (P < 0.001), taking into account food wastage due to preparation and cooking. The cost per unit of energy increased with decreasing fat energy percentage of the diet for all children as one group (r = -0.37, P < 0.001), as well as for the group of children without familial hypercholesterolaemia (r = -0.35, P < 0.001). Stepwise multiple regression analysis showed that the differences in cost per MJ between the groups could be explained primarily by differences in percentage of energy from fat. We conclude that a reduction of dietary fat from 35 E% to 25 E% may increase food costs by 10-20% for Danish children.

Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.

Immunolymphoscintigraphy for metastatic sentinel nodes: test of a model.

Aim. To develop a method and obtain proof-of-principle for immunolymphoscintigraphy for identification of metastatic sentinel nodes. Methods. We selected one of four tumour-specific antibodies against human breast cancer and investigated (1), in immune-deficient (nude) mice with xenograft human breast cancer expressing the antigen if specific binding of the intratumorally injected, radioactively labelled, monoclonal antibody could be scintigraphically visualized, and (2) transportation to and retention in regional lymph nodes of the radioactively labelled antibody after subcutaneous injection in healthy rabbits. Results and Conclusion. Our paper suggests the theoretical possibility of a model of dual isotope immuno-lymphoscintigraphy for noninvasive, preoperative, malignant sentinel node imaging.

Single-transverse-spin asymmetries of identified charged hadrons in polarized pp collisions at sqrt[s]=62.4 GeV.

The first measurements of xF-dependent single-spin asymmetries of identified charged hadrons, pi+/-, K+/-, and protons, from transversely polarized proton-proton collisions at 62.4 GeV at RHIC are presented. Large asymmetries are seen in the pion and kaon channels. The asymmetries in inclusive pi+ production, AN(pi+), increase with xF from 0 to approximately 0.25 and AN(pi-) decrease from 0 to approximately -0.4. Observed asymmetries for K- unexpectedly show positive values similar to those for K+, increasing with xF, whereas proton asymmetries are consistent with zero over the measured kinematic range. Comparisons of the data with predictions of QCD-based models are presented.