The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins.

BACKGROUND AND OBJECTIVES: Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. MATERIALS AND METHODS: The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function. RESULTS: Testisin surface expression increased significantly in capacitated spermatozoa (p < 0.001), increased further following acrosome reaction (p < 0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida-binding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex. CONCLUSION: Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.

Multiple forms of redox activity in populations of human spermatozoa.

In this study we have examined the biochemical attributes of the redox systems that regulate human sperm function using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt (WST-1), lucigenin and luminol-peroxidase as probes. WST-1 was readily reduced by human spermatozoa in the presence of an intermediate electron acceptor (IEA) or NAD(P)H. The IEA-mediated activity resembled a previously described trans-membrane NADH oxidase in being inhibited by capsaicin, superoxide dismutase (SOD) and N-ethyl maleimide, but differed in its sensitivity to p-chloromercuriphenylsulphonic acid (pCMBS). The NAD(P)H-induced WST-1 reduction resembled the superficial oxidase described previously, in its sensitivity to pCMBS, but differed in its suppression by capsaicin. Lucigenin was reduced by human spermatozoa in a manner that could be inhibited by SOD and stimulated by NAD(P)H or 12-myristate, 13-acetate phorbol ester. A23187 also stimulated human spermatozoa via a diphenylene iodonium-sensitive pathway detectable with luminol-peroxidase but not lucigenin. Defective sperm populations recovered from the low-density region of Percoll gradients were characterized by high levels of redox activity that was only discernable with lucigenin. We conclude that human spermatozoa possess multiple plasma membrane redox systems that are involved to varying extents in the physiological control and pathological disruption of sperm function. Their distinct pharmacological profiles should significantly assist attempts to resolve and characterize these systems.

Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer.

This paper describes the development of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 from a lead series of 4-anilinoquinazoline compounds. ZD1839 has suitable properties for use as a clinically effective drug and shows activity against human tumours. In particular, the use of pharmacokinetic data in the development of ZD1839 is discussed.

Utility of tests for circulating melanoma cells in identifying patients who develop recurrent melanoma.

Prospective studies were carried out on 186 patients with AJCC stage I (13), II (76) and III (97 patients) melanoma before and after surgical removal of their tumour. The goal was to determine if PCR tests on blood samples for MART-1 and tyrosinase were predictive of recurrence of melanoma in a 2-year follow-up period. (PCR assays for MUC-18, p97 and gp100 were positive in blood samples from normal subjects and excluded from the study.) PCR tests for MART-1 and tyrosinase were most commonly positive in the first 3 months following surgical removal of melanoma, and three tests over this period gave maximum sensitivity in the identification of patients who subsequently developed recurrences. Positive tests for the first time in the second year of follow-up had similar predictive power. The tests identified 68.5% of patients who developed recurrences in the 2-year follow-up period. Assays for MART-1 were mainly positive in patients with locoregional recurrences, whereas tyrosinase was detected in blood samples from patients with both locoregional and disseminated recurrences. (Positivity rate for tyrosinase in 48 patients with disseminated melanoma was 60.4% compared to 14.6% for MART-1.) Tests for MART-1 and tyrosinase were strongly predictive of disease-free survival (DFS) and were more powerful predictors of DFS than lymph node status or thickness of the primary melanoma. (Hazard ratios by Cox analysis were 2.97 in patients with disseminated recurrences and 2.93 in those with locoregional recurrences.) These results indicate that PCR tests for MART-1 and tyrosinase are powerful prognostic indicators, but their practical utility for selecting patients for adjuvant therapy is limited by the high false-negative rate of approximately 30%. Intermittent shedding of melanoma cells into the circulation would appear to be the most likely explanation for the latter.

MART-1 is expressed less frequently on circulating melanoma cells in patients who develop distant compared with locoregional metastases.

PURPOSE: Polymerase chain reaction (PCR) with tyrosinase and with MART-1 permits detection of small numbers of circulating melanoma cells (CMCs) in patients who have undergone surgical resection of localized disease. In a previous study, we showed that PCR with MART-1 had sensitivity and specificity similar to those of PCR with tyrosinase in terms of detection of CMCs but that PCR with MART-1 seemed to identify a different but overlapping subgroup of patients. In the current study, we examined the utility and prognostic significance of PCR with tyrosinase and with MART-1. PATIENTS AND METHODS: We analyzed the prognostic significance of the patterns of expression of tyrosinase and MART-1 in 186 patients followed sequentially before and after surgical removal of American Joint Committee on Cancer stage I, II, or III melanoma. RESULTS: PCR with tyrosinase and with MART-1 in the first 3 months after surgery identified 68.5% of 73 patients who developed recurrence in the 2-year period after surgery. Approximately 35% of patients with positive tests remained disease-free at 2-year follow-up. We found that patients with disseminated recurrence had a significantly lower incidence of MART-1-positive CMCs (16%) than of tyrosinase-positive CMCs (63%). Patients with locoregional metastases had CMCs that expressed tyrosinase and MART-1 at similar rates. These differences in expression of the markers in patients with disseminated recurrence were also associated with a much lower disease-free survival, in those who had CMCs that were positive for tyrosinase but negative for MART-1. The reverse applied in those with locoregional disease. CONCLUSION: These findings suggest that PCR with MART-1 and with tyrosinase identifies subgroups of patients who develop disseminated or locally recurrent metastases. We hypothesize that immune responses against MART-1 may reduce the establishment of disseminated metastases.

Evaluation of S-100beta assays for the prediction of recurrence and prognosis in patients with AJCC stage I-III melanoma.

We performed prospective serial studies on 266 melanoma patients undergoing surgery for high risk primary or lymph node metastases to assess the predictive value for recurrence of melanoma of S-100beta detection in the serum using immunoradiometric assay (IRMA) or Immunoluminometric assay (LIA-mat). The tests for S-100beta were most frequently positive in the first 3 months after surgery. Results of the assays did not show a strong correlation with clinical stage of the disease. Studies on a cohort of 147 patients who had been followed for a minimum of 2 years post-surgery were carried out to assess the sensitivity of the assays for identifying patients who develop recurrence of their melanoma. The LIA-mat and IRMA assays detected S-100beta in the serum of 47.5% and 23%, respectively, of the subset of 61 patients who developed recurrence of disease. By Kaplan Meler analysis, patients positive for S-100beta by LIA-mat assay in the first 3 months post-surgery were shown to have an approximately 2.7 times shorter disease-free survival (DFS) period than patients negative for S-100beta. This was significant by log rank analysis. Multivariate analysis indicated that the presence of S-100beta was an independent prognostic determinant of DFS, and was independent of tumour thickness and lymph node status. This prospective analysis in a large number of patients suggests that assays for S-100beta in patients following surgical resection of AJCC stage I-III melanoma are of limited value for identifying patients who will develop recurrence of their disease. The results of the LIA-mat assays, but not the IRMA assays, do however provide an additional measure of prognosis that is independent of existing measures.

Polymerase chain reaction detection of melanoma cells in the circulation: relation to clinical stage, surgical treatment, and recurrence from melanoma.

PURPOSE: The detection of melanoma cells in the circulation by polymerase chain reaction (PCR) assays has been shown by several investigators to correlate with the stage of the disease and possibly with prognosis. PATIENTS AND METHODS: We performed prospective studies on 276 patients with primary melanoma and regional lymph node (LN) metastases to assess the predictive value of PCR detection of tyrosinase and melanoma antigen recognized by T cells-1 (MART-1) in the blood for recurrence of melanoma. RESULTS: PCR tests for gp 100, Muc-18, and p97 reacted with RNA in blood from healthy subjects and were considered unsuitable for patient monitoring. The tests were most frequently positive in the first 3 months after surgery. There were 47 recurrences in 123 patients who had been followed up for 18 months. Assays within 3 months of surgery predicted recurrence from melanoma in 66% of the latter (tests for tyrosinase alone detected 51% and MART-1 alone 21% of the patients). Hence, 34% of recurrences were not predicted by tests in the early postoperative period. This did not appear to be because of marker-negative melanoma because summation of tests over the first year identified 89% of those with recurrent disease. CONCLUSION: Positive tests were recorded in 35% of patients who remained disease free, but it is too early to assess whether these represent false-positive results. The false-negative results raise the question of whether the assays will provide a reliable basis for selection of patients for adjuvant therapy.

Detection and quantitation of melanoma cells in the circulation of patients.

A method for the quantitation of tyrosinase mRNA in the bloodstream of melanoma patients has been developed by using competitive polymerase chain reaction (PCR) with a heterologous DNA (PCR MIMIC) as an internal standard. The method was validated by demonstration of similar amplification efficiencies for both molecules and by accurate quantitation of an artificial fourfold difference in the level of tyrosinase mRNA. The ratio of amplified target to amplified standard (At/As ratio) was determined for a range of melanoma patients who had previously tested positive for tyrosinase. Sequential samples were also analysed to examine the level of tyrosinase in individual patients over time. When tyrosinase levels in melanoma cell lines were compared for a constant amount of total RNA, or for a constant number of cells, tyrosinase mRNA was found to vary more than a thousand-fold between cell lines. Because of this, the At/As ratio of patients was compared with the At/As ratios obtained when 10-fold serial dilutions of cells from a control melanoma cell line (MM200) were added to 2 ml of packed blood. An 'MM200 equivalence' value was thus calculated, giving the equivalent number of MM200 cells in the bloodstream of melanoma patients. Prolonged follow-up will be needed to determine the prognostic significance of the detection and levels of tyrosinase mRNA in the bloodstream of melanoma patients.

Spontaneous thrombosis of an unruptured anterior communicating artery aneurysm. An unusual cause of ischemic stroke.

BACKGROUND: Stroke caused by spontaneous thrombosis of an unruptured intracranial aneurysm is a rare event. CASE DESCRIPTION: A 66-year-old woman experienced a transient ischemic attack and cerebral infarctions due to spontaneous thrombosis of an unruptured anterior communicating artery aneurysm. Extension of thrombus into both anterior cerebral arteries and the left middle cerebral artery, resulting in ischemic infarction in all three vascular territories, was diagnosed by CT scanning, MRI, and cerebral angiography and confirmed at autopsy. CONCLUSIONS: This case illustrates a rare complication of an unruptured saccular aneurysm with neuroimaging and pathological correlation. Morphological and hemodynamic factors that may have precipitated aneurysm thrombosis are discussed with reference to experimental models.

Effect of short-term fasting/refeeding on epidermal growth factor content in the gastrointestinal tract of suckling rats.

Epidermal growth factor (EGF) is trophic for varying regions of the developing gastrointestinal tract (GIT) of suckling rats. The presence of large amounts of EGF in milk from various species, combined with low production of EGF by suckling animals, led to speculation that milk is a major source of EGF for suckling rats. We report that short-term fasting (8 hr) of 12-day-old suckling rats resulted in a significant decrease in the levels of immunoreactive EGF (irEGF) in the GIT. Pups refed by lactating mothers for 1 to 4 hr exhibited an increase in irEGF to original levels, whereas pups fed a rat milk substitute by gastric gavage did not have an increase in irEGF content. The irEGF levels in the GIT of pups that were manually fed normal rat milk, or rat milk substitute supplemented with EGF, returned to the prefasted levels. Fasted suckling rats refed 2 ml of rat milk in 2 h exhibited significantly higher level of irEGF in the GIT than did those refed with 0.5 ml in 45 min. Since rat milk irEGF exists in three distinct forms (A, B, and C; C is equal to authentic submandibular gland EGF, the irEGF forms in the GIT were characterized by native polyacrylamide gel electrophoresis. In the stomach luminal contents of the fed suckling rats, only the larger form, Peak B, was observed. Both the luminal content and the mucosa scrapings of all other segments of all groups contained only Form D (comigrating with desarginyl EGF), a metabolic derivative of EGF. All forms were immunoreactive, exhibited receptor binding, and stimulated DNA synthesis in growth-arrested fibroblasts. The rapid changes in EGF within the GIT of suckling rats suggest the EGF can acutely modify some GIT functions of suckling rats.

Pancreatic secretory trypsin inhibitor stimulates the growth of rat pancreatic carcinoma cells.

Pancreatic secretory trypsin inhibitor was examined for growth-promoting activity on five cell lines using standard cell culture techniques. One cell line, AR4-2J, derived from a rat pancreatic acinar cell carcinoma, responded with significantly increased incorporation of [3H]thymidine and colony formation. Pancreatic secretory trypsin inhibitor stimulated the incorporation of [3H]thymidine in liquid culture; the maximal increase was 61 +/- 10% above control (P less than 0.001) and was seen at a concentration of 10(-9) mol/L. Using a soft agarose clonogenic assay, pancreatic secretory trypsin inhibitor also consistently stimulated (3 assays) colony formation: the peak activity occurred at a concentration of 10(-10) mol/L which caused a 150 +/- 55% (mean +/- SE, P less than 0.05) increase above control. Aprotinin had no effect on the growth of AR4-2J cells and pancreatic secretory trypsin inhibitor did not bind to the epidermal growth factor receptor. AR4-2J cells were shown to produce pancreatic secretory trypsin inhibitor. The study raises the possibility that pancreatic secretory trypsin inhibitor provides autocrine stimulation of tumor cell growth.

The use of polymyxin B and C3H/HeJ mouse spleen cells as criteria for endotoxin contamination.

We have performed experiments designed to evaluate the potential contribution of endotoxin contamination to lymphocyte reponses. Saline and EDTA extracts of 4 different strains of gram negative bacteria were examined for their capacity to initiate mitogenic responses in murine spleen cells. As compared to phenol extracts of these bacteria which contain primarily lipopolysaccharide-LPS, these saline and EDTA extracts were significantly less active in this assay. The mitogenic activity which was present was also manifest in spleen cells from the C3H/HeJ mouse, whereas phenol-extracted LPS preparations were inactive. In addition, mitogenic activity of saline and EDTA extracts was not blocked by polymyxin B, an agent known to abrogate LPS mediated responses. We conclude that LPS contamination may not normally be as significant a problem as had earlier been assumed. However, when endotoxin contamination is present, neither the use of C3H/HeJ spleen cells nor polymyxin B is an appropriate test to evaluate this possibility.

Role of complement in endotoxin initiated lethality in mice.

We have examined the role of complement in eliciting a lethal response in C3H/HeJ and C3H/St mice. The results reported here indicate that endotoxin-initiated complement activation, leading to significant drops in circulating C3 levels, is not sufficient to cause lethality. The complement system in both strains was demonstrated to be responsive in vitro to activation both by E. coli 0111:B4 LPS I, an alternative pathway activator in other systems, as well as S. minnesota R595 LPS, which activates almost exclusively the classical pathway. In vivo injection of high (lethal) doses of E. coli 0111:B4 LPS I and S. minnesota R595 LPS causes a significant decrease in the circulating C3 levels of both strains after 4 hr. In contrast, circulating C3 levels were not significantly different from normal values in either strain following injection with minimal (lethal) amounts of E. coli 0111:B4 LPS II, a weakly anticomplementary LPS preparation. In all cases, lethality was observed in only the C3H/St mice, indicating that neither complement activation, nor the lack of it, is responsible for lethality in mice.

Two distinct mechanisms for the initiation of mast cell degranulation. II. A specific inhibition of amine release by serum proteins.

Our experiments have provided additional data in support of the concept that different mast cell activators follow distinct biochemical path-ways in the initiation of secretion and degranulation. To do this we have taken advantage of the observation that some serum proteins, and albumins in particular, have the capacity to inhibit selectively the release of amines from rat peritoneal mast cells initiated by some, but not all, stimuli. We show that the relative inhibition of release obtained is independent of the concentration of activator but dependent upon the concentration of albumin, indicating that the inhibitory process does not involve a direct activator--inhibitor interaction. Finally, our data demonstrate that the inhibition does not interfere with the ability of the acitivator to interact with the mast cell. Thus, cells incubated with activator in the presence of inhibitor become increasingly unresponsive, or desensitized, to subsequent challenge with activator in the absence of inhibitor. These combined data therefore provide evidence, that, in addition to a selectivity in the activation/desensitization process initiated by different mast cell stimuli, at least one of the biochemical steps subsequent to the activation step is also not shared by all mast cell stimuli.