Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.

Background: Studies on the diversity of carbohydrate-binding proteins (lectins) are important in glycobiology. Results: A lectin having a novel primary structure was isolated from a mussel and found to have a globotriose-dependent cytotoxicity on Burkitt lymphoma cells. Conclusion: A new primary structure quite distinct from known lectin is described. Significance: Discovery of similar lectin structures from vertebrates will lead to progress in medical sciences.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Sialic acid binding lectin (SBL) isolated from Rana catesbeiana oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity. However, the mechanism of antitumor effects of SBL is unclear to date and the validity for human leukemia cells has not been fully studied. We report here that SBL shows cytotoxicity for some human leukemia cell lines including multidrug-resistant (MDR) cells. The precise mechanisms of SBL-induced apoptotic signals were analyzed by combinational usage of specific caspase inhibitors and the mitochondrial membrane depolarization detector JC-1. It was demonstrated that SBL causes mitochondrial perturbation and the apoptotic signal is amplified by caspases and cell death is executed in a caspase-dependent manner. The efficacy of this combinational usage was shown for the first time, to distinguish the apoptotic pathway in detail. SBL selectively kills tumor cells, is able to exhibit cytotoxicity regardless of P-glycoprotein expression and has potential as an alternative to conventional DNA-damaging anticancer drugs.

ABSTRACT-Wereported previously that cilnidipine inhibited increases in blood pressure and plasma norepinephrine (NE) level in response to cold stress in spontaneously hypertensive rats (SHRs). In the present study, we investigated the effect of cilnidipine on sympathetic neurotransmission and subsequent vasoconstriction in SHRs. In pithed SHRs, electrical sympathetic nerve stimulation (ESNS) elevated blood pressure, and this pressor response was abolished by guanethidine. Cilnidipine at 10 tug/kg, i.v. and phentolamine at 1 mg/kg, i.v. suppressed the pressor response to ESNS by 28 ± 6% and 67 ± 3 %, respectively. Neither nifedipine nor nicardipine inhibited it. The pressor response to exogenous NE was not influenced by cilnidipine. a,~-Methylene ATP inhibited the pressor response to ESNS in the presence or absence of phentolamine. Cilnidipine also attenuated the phentolamine-resistant pressor response to ESNS. In SHR mesenteric vasculatures preloaded with [3H]-NE, cilnidipine (10-7 M) as well as w-conotoxin significantly inhibited the 3H overflow evoked by periarterial nerve stimulation. In radioligand binding experiments, cilnidipine inhibited [1251]-w-conotoxin binding to rat synaptosomes, but it did not inhibit [3H]-prazosin binding to rat cortex membranes. These results suggest that cilnidipine may reduce electrically stimulated NE release from the sympathetic nerve endings of SHR vasculatures probably through its N-type Ca channel blocking action and that cilnidipine may also inhibit the vasoconstriction induced by ATP released concomitantly during nerve stimulation.Keywords: Cilnidipine, Pithed spontaneously hypertensive rat, Norepinephrine release, a,~-Methylene ATP, w-ConotoxinCilnidipine is a new 1,4-dihydropyridine (DHP) derivative calcium (Ca) channel blocker that has a slow-onset and long-lasting hypotensive action (1). Cilnidipine has been also reported to inhibit the pressor response to acute cold stress in spontaneously hypertensive rats (SHRs) (2). It has been suggested that the inhibition of the pressor response by cilnidipine may be due to reduced sympathetic nerve activity, because cilnidipine attenuated the cold stress-induced increase in plasma norepinephrine (NE) concentration in SHRs (2).In the present study, in order to further elucidate the mechanism of the inhibition by cilnidipine of cold stressinduced elevation in blood pressure, we investigated the effects of cilnidipine on both the pressor response to electrical sympathetic nerve stimulation (ESNS) in pithed SHR and the 3H overflow evoked by periarterial nerve stimulaton (PNS) from perfused SHR mesenteric vasculatures preloaded with [3H]-NE. In some experiments, the effects of cilnidipine on [3H]-prazosin and [1251]-wconotoxin (w-CTx) bindings to rat cortex membranes and rat synaptosomes, respectively, were also studied. MATERIALS AND METHODS Animals usedMale 18-to 27-week-old SHRs (Charles River Japan, Yokohama) weighing 322-420g were used. Male Sprague-Dawley (SD) rats (Charles River Japan) weighing 160-180 g were also used for bi...

Background: Despite crucial roles of polysialic acid (polySia) in neural functions, the enzyme involved in degradation of polysialic acid in its physiological turnover remains uncertain. Results: Sialidase NEU4 catalytically degrades polySia and negatively regulates neurite outgrowth of hippocampal neurons. Conclusion: Sialidase NEU4 is probably the major degradation enzyme for polySia in vertebrate. Significance: The findings contribute to elucidation of the physiological turnover of polySia.

Malignant mesothelioma is a highly aggressive tumor with poor prognosis. An effective drug for treatment of malignant mesothelioma is greatly needed. Sialic acid-binding lectin (SBL) isolated from oocytes of Rana catesbeiana is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity, so it could be developed as a new type of anticancer drug. The validity of SBL for treatment of malignant mesothelioma was assessed using three malignant mesotheliomas and a non-malignant mesothlial cell line. Effectiveness of combinatorial treatment of SBL and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was also elucidated and characterized. SBL induced tumor-selective cytotoxicity that was attributed to induction of apoptosis. Combinatorial treatment of SBL and TRAIL showed synergistic apoptosis-inducing effect. Additional experiments revealed that Bid was the mediating molecule for the synergistic effect in SBL and TRAIL. These results suggested that SBL could be a promising candidate for the therapeutics for malignant mesothelioma. Furthermore, the combinatorial treatment of SBL and TRAIL could be an effective regimen against malignant mesothelioma.

ABSTRACT-We investigated the effect of cilnidipine on cardiovascular and neuroendocrine responses to acute cold stress in conscious and unrestrained or moderately restrained spontaneously hypertensive rats (SHRs). Acute cold stress significantly increased mean blood pressure without marked change in heart rate. The acute cold stress-induced elevation in blood pressure was almost abolished by 1 mg/kg, p.o. of prazosin. The cold stress also elevated plasma norepinephrine and epinephrine levels. Cilnidipine at 3 mg/kg, p.o. significantly inhibited the pressor response to acute cold stress. Although 3 mg/kg, p.o. of nifedipine, nicardipine or manidipine lowered mean blood pressure to a similar extent as cilnidipine, none of these three drugs affected the pressor response. Cilnidipine also reduced the cold stress-induced increment in plasma norepinephrine but not the epinephrine level. These findings suggest that acute cold stress may induce the elevation in blood pressure due to an enhanced activation of the sympathoadrenal system in SHRs and that cilnidipine may suppress the pressor response by inhibiting the sympathetic nerve activity.