A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin.

PURPOSE: To determine whether the 12-lipoxygenase pathway of arachidonic acid metabolism is present in the human lens and whether 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) plays a role in regulating proto-oncogene expression and DNA synthesis in human lens epithelial cells (HLECs). METHODS: Second- and third-passage primary cultures of HLECs were used for analysis. Human cataract epithelia were obtained from surgery. 12-lipoxygenase mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR product was sequenced. The 12-lipoxygenase protein was detected by immunoblotting. 12(S)-HETE was detected in HLEC-conditioned medium by radioimmunoassay. For studies of growth factor-induced mitogenesis, HLECs were serum starved, then stimulated with 15 ng/ml epidermal growth factor (EGF) and 1 microgram/ml insulin or with 0.3 microM 12(S)-HETE. The 12-lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10 microM) was used to block endogenous 12-lipoxygenase activity. Expression of c-fos mRNA was determined by RT-PCR, and DNA synthesis was measured by 3H-thymidine incorporation. RESULTS: 12-lipoxygenase mRNA and protein were detected in HLECs and in human cataract tissues. 12(S)-HETE was released into the medium by HLECs in the presence of EGF-insulin. Stimulation of c-fos mRNA expression and DNA synthesis by EGF-insulin was inhibited when the 12-lipoxygenase pathway was blocked by CDC. This inhibition was reversed completely by exogenously added 12(S)-HETE. However, exogenous 12(S)-HETE was unable to stimulate HLEC DNA synthesis in the absence of growth factors. CONCLUSIONS: The 12-lipoxygenase pathway of arachidonic acid metabolism is present in human lens epithelial cells. 12(S)-HETE does not stimulate HLEC DNA synthesis in the absence of growth factors but enables the cellular response to EGF and insulin.

The rabbit lens epithelial cell line N/N1003A requires 12-lipoxygenase activity for DNA synthesis in response to EGF.

PURPOSE: Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics. METHODS: Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA). RESULTS: 12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors. CONCLUSIONS: N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.

Camel trypanosomosis in Rajasthan, India.

Blood samples from 240 camels (Camelus dromedarius) were examined for trypanosome infection. Of these, 18 (7.50%) were found to be infected using the wet blood Giemsa stain technique, while 76 (31.66%) camels were found to be positive for Trypanosoma evansi antigen using the double antibody sandwich enzyme-linked immunosorbent assay (ELISA). The latter was found to be a more useful method for the detection of current infection.

12(S)-hydroxyeicosatetraenoic acid regulates DNA synthesis and protooncogene expression induced by epidermal growth factor and insulin in rat lens epithelium.

Neonatal rat lens epithelium has a high 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] synthetic capacity, which decreases as epithelial cell proliferation decreases with age. To determine whether products of the 12-lipoxygenase pathway are involved in lens cell proliferation, we measured the effect of 12-lipoxygenase inhibitors on endogenous 12-HETE production, epidermal growth factor/insulin-stimulated DNA synthesis and protooncogene expression in cultured neonatal rat lens epithelial cells. Incubation of neonatal rat lenses in epidermal growth factor plus insulin, which stimulated endogenous 12-HETE production 8- to 10-fold, also produced a transient induction of c-fos and c-myc mRNAs after 2 to 3 h, followed by a round of DNA synthesis approximately 20 h later. The lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, strongly inhibited both the endogenous 12-HETE synthesis and growth factor-stimulated DNA synthesis with a half-maximal inhibition between 10 and 20 microM. Cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (10 microM) also inhibited the expression of c-fos and c-myc mRNA and, to a lesser extent, c-jun mRNA. The inhibitory effects of cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate on protooncogene expression and DNA synthesis were prevented by 0.3 microM 12(S)-HETE but not by equivalent concentrations of either 5(S)-HETE or 15(S)-HETE. These findings suggest that endogenously synthesized 12(S)-HETE may mediate epidermal growth factor/insulin-stimulated DNA synthesis in neonatal rat lens epithelial cells by regulating protooncogene expression.

Impact of geographic proximity to cardiac revascularization services on service utilization.

BACKGROUND: In a highly competitive health care environment, even microgeographic differences in availability of tertiary services might affect access to care. OBJECTIVES: To study the impact of (1) geographic distance from patient's residence to cardiac revascularization services and (2) the availability of cardiac revascularization services at the hospital nearest the patient's residence on utilization of these services in a geographically small, densely populated area. METHODS: Historical cohort study of 55,659 New Jersey residents hospitalized between 1992 and 1996 with primary diagnosis of acute myocardial infarction (AMI). MAIN STUDY OUTCOMES: Use of percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass graft surgery (CABG) within 90 days of initial hospitalization for AMI and in-hospital mortality. Distance from patient's residence to nearest hospital with cardiac revascularization services (PTCA and CABG) was a straight-line distance in miles, categorized as 0 to <2, 2 to <5, 5 to <10, 10 to <15, 15 to <20, 20 to <25, > or =25 miles. Adjusted odds of PTCA or CABG use at each distance category were compared with odds at > or =25 miles. RESULTS: A strong linear decline in adjusted odds ratios for PTCA use was found with increasing distance of this service from the patient's residence (p <0.05). Adjusted odds of PTCA use were 2.4, 2.1, 1.8, 1.5, 1.3, and 1.0 times higher for each increasing distance category in comparison with > or =25 for patients aged <65 and 3.1, 2.7, 2.2, 1.9, 1.7, and 1.1 for patients aged > or =65. Use of CABG was also higher for patients residing closer to cardiac revascularization services. The availability of these services at the hospital nearest to the patient's residence also increased utilization. In-hospital mortality was not associated with distance from services. CONCLUSION: Even across a relatively small geographic area, shorter distance to services and availability of services at the nearest hospital were strongly related to increased utilization of cardiac revascularization services.