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AIM: The aim of this study is to commission and validate the portal dosimetry (PD) system using an indirect method for flattening filter free (FFF) photon beam of the upgraded c-series linear accelerator. BACKGROUND: Varian Medical System clinacs with amorphous-silicon portal imager panel (aSi-1000) do not have PD for FFF beams. Recently, our c-series linear accelerator was upgraded to deliver 6MV FFF (6MVFFF) photon beam with the highest dose rate of 1400 monitor unit (MU)/min. The study, therefore, focuses on the commissioning and validation of PD for the 6MVFFF beam. MATERIALS AND METHODS: An indirect method was implemented to predict the portal dose for FFF beam in Eclipse as the treatment planning system does not have direct prediction algorithm for FFF beam (version. 11). Dosimetrical characteristics of aSi-electronic portal imaging device (EPID) were evaluated for 6MVFFF beam and validation of PD for 6MVFFF beam was performed for open fields along with pretreatment quality assurance of intensity-modulated radiation therapy (IMRT), volumetric-modulated arc therapy (VMAT), and stereotactic radiosurgery (SRS) techniques for 30 patients planned with 6MVFFF beam. RESULTS: ASi-EPID saturates between 100 and 130 cm source to detector distance (SDD) for 6MVFFF beam and resolved at more than 140 cm SDD. The squared correlation coefficient (R2) for MU linearity was found to be 1 (R2 = 1), and instantaneous dose response linearity at different SDD's was found to be 0.999 (R2 = 0.999) for the 6MVFFF beam. Maximum gamma area index (GAI) for 3% dose difference and 3 mm distance-to-agreement criteria for IMRT, VMAT, and SRS/stereotactic radiotherapy plans was 97.9% ± 0.3%, 96.3% ± 0.5%, and 98.2% ± 0.2%, respectively. CONCLUSION: The results reveal that this novel method can be used to commission portal dosimetry for 6MVFFF beam as it is a convenient, faster, and accurate method.

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AIM: Purpose of this study is to dosimetrically compare head and neck (H and N) cancer patients planned with multivendor volumetric modulated arc therapy (VMAT) technology. VMAT treatment planning can be done using biological (treatment planning system [TPSB]: Monaco) or physical (TPSP: Eclipse)-based cost function optimization techniques. Planning and dosimetric comparisons were done in both techniques for H and N cases. MATERIALS AND METHODS: Twenty H and N patients were retrospectively selected for this study. VMAT plans were generated using TPSP (V11.0) and TPSB (V3.0) TPS. A total dose of 66 Gy (planning target volume 1 [PTV1]) and 60 Gy (PTV2) were prescribed to primary and nodal target volumes. Clinical planning objectives were achieved by both the optimization techniques. Dosimetric parameters were calculated for PTVs, and quantitative analyses were performed for critical organs. Monitor units were compared between two TPSs, and gamma analysis was performed between I'matriXX measured and TPS calculated. RESULTS: Clinically, acceptable VMAT plans showed comparable dose distributions between TPSB and TPSP optimization techniques. Comparison of mean dose, homogeneity index, and conformity index for PTV1 showed no statistical difference (P - 0.922, 0.096, and 0.097); however, in PTV2 statistically significant difference was observed (P - 0.024, 0.008, and 0.002) between TPSB and TPSP. TPSB optimization showed statistically significant superiority for spinal cord and brainstem (D1% P - 0.0078, 0.00002) whereas improved parotid sparing was observed in TPSP optimization (mean dose P - 0.00205). Gamma analysis illustrated that both systems could produce clinically deliverable plans. CONCLUSION: VMAT plans by TPSP and TPSB offered clinically acceptable dose distributions. TPSB-based optimization showed enhanced sparing of serial organs whereas TPSP-based optimization showed superior sparing of parallel organs.

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In order to protect the low noise amplifier (LNA) in the receive arm of a pulsed 250 MHz EPR bridge, it is necessary to install as much isolation as possible between the power exciting the spin system and the LNA when high power is present in the receive arm of the bridge, while allowing the voltage induced by the magnetization in the spin sample to be passed undistorted and undiminished to the LNA once power is reduced below the level that can cause a LNA damage. We discuss a combination of techniques to accomplish this involving the power-routing circulator in the bridge, a second circulator acting as an isolator with passive shunt PIN diodes immediately following the second circulator. The low resistance of the forward biased PIN diode passively generates an impedance mismatch at the second circulator output port during the high power excitation pulse and resonator ring down. The mismatch reflects the high power to the remaining port of the second circulator, dumping it into a system impedance matched load. Only when the power diminishes below the diode conduction threshold will the resistance of the PIN diode rise to a value much higher than the system impedance. This brings the device into conduction mode. We find that the present design passively limits the output power to 14 dBm independent of the input power. For high input power levels the isolation may exceed 60 dB. This level of isolation is sufficient to fully protect the LNA of pulse EPR bridge.

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The authors have obtained spectral-spatial EPR images of a phantom significantly larger than those previously obtained. Images of a homogeneous phantom 4.2 cm in diameter and 6.5 cm in length with B1 equivalent to that used for smaller samples give a similar linewidth resolution both with linewidth population distributions of width 0.1 microT. Spatial resolution appeared to have modest degradation. Images of the large homogeneous phantom provide maps of the magnetic field of a partially shimmed magnet.

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The major cellular pathway for uptake of the vitamin folic acid, including its absorption in the intestine, is via a plasma membrane carrier system, the reduced folate carrier (RFC). Very little is known about the mechanisms that control intracellular trafficking and plasma membrane targeting of RFC. To begin addressing these issues, we used Xenopus oocyte as a model system and examined whether the signal that targets the protein to the plasma membrane is located in the COOH-terminal cytoplasmic tail or in the backbone of the polypeptide. We also examined the role of microtubules and microfilaments in intracellular trafficking of the protein. Confocal imaging of human RFC (hRFC) fused to the enhanced green fluorescent protein (hRFC-EGFP) showed that the protein was expressed at the plasma membrane, with expression confined almost entirely to the animal pole of the oocyte. Localization of hRFC at the plasma membrane was not affected by partial or total truncation of the COOH-terminal tail of the polypeptide, whereas a construct of the cytoplasmic tail fused to EGFP was not found at the plasma membrane. Disruption of microtubules, but not microfilaments, prevented hRFC expression at the plasma membrane. These results demonstrate that the molecular determinant(s) that directs plasma membrane targeting of hRFC is located within the backbone of the polypeptide and that intact microtubules, but not microfilaments, are essential for intracellular trafficking of the protein.

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Previous studies from our laboratory have demonstrated the existence of a folate transporter in the human colonic apical membranes. The current studies were undertaken to examine the possible presence and function of a folate carrier in the human colonic basolateral membrane vesicles (BLMV). BLMV were purified from mucosal scrapings of colons of organ donors by a Percoll-density gradient centrifugation technique, and uptake studies were performed using a rapid filtration technique. Our results on [(3)H]Pte-Glu uptake are summarized as follows: 1) uptake was sensitive to osmolarity of the incubation medium; 2) Na(+) removal from the incubation medium did not affect folate uptake into BLMV; 3) uptake was significantly increased with decreasing incubation buffer pH from 8 to 4; 4) uptake demonstrated saturation kinetics with an apparent Michaelis constant of 9.6 +/- 0.48 microM and a maximal velocity of 8.10 +/- 0.36 pmol x mg protein(-1) x 10 s(-1); 5) uptake was markedly inhibited by the structural analog methotrexate (inhibitory constant = 8.28 +/- 1.0 microM); 6) uptake into BLMV demonstrated a trans-stimulation phenomenon; 7) anion exchange inhibitors DIDS and SITS significantly inhibited folate uptake; and 8) uptake was potential-insensitive, as voltage clamping of vesicles or making them inside positive with K(+)/valinomycin failed to influence folate uptake. Western blot analysis using purified human colonic basolateral membrane preparations and specific polyclonal antibodies against the human reduced folate carrier (hRFC) has shown expression of the hRFC protein at this membrane domain. These data demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral, carrier-mediated mechanism for folate transport across the human colonic basolateral membranes.

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Thiamine (vitamin B(1)) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) Na(+) independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a Ca(2+)/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes.

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Folate is an essential micronutrient that, in mammals, must be obtained from exogenous sources via intestinal absorption. Previous studies have characterized different aspects of the mechanism of the intestinal folate uptake process. Much less, however, is known about regulation of this process. In this study, we examined the effect of dietary folate deficiency on intestinal folate uptake using the rat as an animal model. The results showed that dietary folate deficiency leads to a significant (P < 0.01) and specific upregulation in the transepithelial transport of folic acid. The upregulation in transepithelial folate transport 1) was found to be due to an induction in carrier-mediated folate uptake across the brush-border membrane (BBM) and was mediated via a significant (P < 0.01) increase in the maximal velocity but not the apparent Michaelis constant of the uptake process, 2) was associated with a marked increase in the steady-state mRNA level of reduced folate carrier-1 and in the level of the expressed protein at the intestinal BBM, and 3) was associated with a marked (>10-fold) increase in the activity of the intestinal BBM form of folate hydrolase. Results of this study demonstrate, for the first time, that dietary folate deficiency leads to a marked upregulation in intestinal folate uptake and in the activity of folate hydrolase. Furthermore, the upregulation in folate uptake is associated with an increase in mRNA and protein levels of folate carrier, suggesting possible involvement of a transcriptional regulatory mechanism(s) in the upregulation.

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Although sphingomyelin (SPH) is a major constituent of all lipoproteins, its physiological function in plasma is not known. In this study, we tested the hypothesis that SPH inhibits lipid peroxidation in low density lipoproteins (LDL) because of its effects on surface fluidity and packing density and that the relative resistance of the buoyant LDL to oxidation, compared with the dense LDL, is partly due to their higher SPH content. Depletion of SPH by treatment with SPHase resulted in shortened lag times and increased rates of oxidation in both LDL subfractions, as measured by the conjugated diene formation in the presence of Cu(2+). Oxidation of LDL by soybean lipoxygenase was similarly stimulated by the degradation of SPH. Oxidation-induced fluorescence decay of diphenylhexatriene-labeled phosphatidylcholine (PC), equilibrated with LDL-PC, was accelerated significantly by the enzymatic depletion of SPH from the lipoprotein. Oxidation of 16:0-18:2 PC in the proteoliposomes was inhibited progressively by the incorporation of increasing amounts of egg SPH into the liposomes. Treatment of SPH-containing proteoliposomes with SPHase reversed the effect of SPH, showing that the presence of intact SPH is necessary for the inhibition of oxidation. Although the incorporation of SPH into the same liposome as the PC (intrinsic SPH) protected the PC against oxidation, the addition of SPH liposomes to PC liposomes (extrinsic SPH) was not effective. Oxidation of 16:0-18:2 PC in liposomes was also inhibited by the incorporation of dipalmitoyl-PC, but not by free cholesterol. These results suggest that SPH acts as a physiological inhibitor of lipoprotein oxidation, possibly by modifying the fluidity of the phospholipid monolayer and thereby inhibiting the lateral propagation of the lipid peroxy radicals.

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To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-14C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.

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Human lecithin-cholesterol acyltransferase (LCAT), which is normally specific for the sn-2 position of phosphatidylcholine (PC), derives a significant percentage of acyl groups from the sn-1 position, when sn-2 is occupied by 18:0, 20:4, or 22:6. We investigated the relative importance of the two acyl groups of PC in determining the positional specificity by first analyzing the cholesteryl esters formed in the presence of symmetric PCs labeled at sn-2. Both human and rat LCATs transferred exclusively the sn-2-acyl group from all symmetric PCs, including 18:0-18:0, and 20:4-20:4, showing that the presence of these fatty acids at sn-2 does not automatically alter the positional specificity. The role of the sn-1-acyl group was then tested by using PCs containing 20:4 or 18:0 at sn-2 and fatty acids of various chain lengths and unsaturation at sn-1. With 20:4 at sn-2 and saturated fatty acids of various chain lengths at sn-1, human and rat LCATs derived, respectively, 5-72% and 1-20% of the total acyl groups from the sn-1 position. However, the chain length of the sn-1-acyl did not correlate with its utilization by either enzyme. Various unsaturated fatty acids at sn-1 also were transferred by human LCAT at a higher rate (5-75% of total) than they were transferred by rat LCAT (0-21%). With sn-2-18:0 PCs, however, rat LCAT exhibited greater alteration in positional specificity (30-95% from sn-1) than human LCAT (15-83% from sn-1). These results show that while the primary determinant of positional specificity is the sn-2-acyl group of PC, the structure of sn-1-acyl significantly modifies it.

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Although dietary trans unsaturated fatty acids (TUFA) are known to decrease plasma HDL, the underlying mechanisms for this effect are unclear. We tested the hypothesis that the decreased HDL is due to an inhibition of lecithin:cholesterol acyltransferase (LCAT), the enzyme essential for the formation of HDL, by determining the activity of purified LCAT in the presence of synthetic phosphatidylcholine (PC) substrates containing TUFA. Both human and rat LCATs exhibited significantly lower activity (-37% to -50%) with PCs containing 18:1t or 18:2t, when compared with the PCs containing corresponding cis isomers. TUFA-containing PCs also inhibited the enzyme activity competitively, when added to egg PC substrate. The inhibition of LCAT activity was not due to changes in the fluidity of the substrate particle. However, the inhibition depended on the position occupied by TUFA in the PC, as well as on the paired fatty acid. Thus, for human LCAT, 18:1t was more inhibitory when present at sn-2 position of PC, than at sn-1, when paired with 16:0. In contrast, when paired with 20:4, 18:1t was more inhibitory at sn-1 position of PC. Both human and rat LCATs, which are normally specific for the sn-2 acyl group of PC, exhibited an alteration in their positional specificity when 16:0-18:1t PC or 16:1t-20:4 PC was used as substrate, deriving 26-86% of the total acyl groups for cholesterol esterification from the sn-1 position. These results show that the trans fatty acids decrease high density lipoprotein through their inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, and also alter LCAT's positional specificity, inducing the formation of more saturated cholesteryl esters, which are more atherogenic.

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PURPOSE: To assess the bioequivalence of nadolol 40mg and 160mg tablets (Zenith-Goldline Pharmaceuticals) using Corgard 40mg and 160mg tablets (Bristol-Meyers Squibb) as reference products, to estimate the effect of food in the gastrointestinal tract on nadolol bioavailability, and to evaluate the effectiveness of standard pharmacokinetic metrics AUCt, AUC infinity, and Cmax in bioequivalence determinations. METHODS: Four bioequivalence studies were conducted as described in the FDA Guidance. Four additional studies of varying designs were conducted to establish bioequivalence of the 40mg tablet in terms of Cmax. RESULTS: Fasted and food-effect studies of the 160mg tablet clearly established bioequivalence and revealed an unexpected reduction in nadolol bioavailability from test and reference products in the presence of food. The food-effect study of the 40mg tablet (80mg dose) revealed a similar reduction in bioavailability from each product. Fasted studies of the 40mg tablet (80mg dose) established bioequivalence in terms of AUCt and AUC infinity. However, Cmax criteria proved extremely difficult to meet in the initial 40mg fasted study because of the large variability, leading to additional studies and ultimately requiring an unreasonable number of subjects. CONCLUSIONS: Final results clearly established bioequivalence of both strengths and characterized an unexpected food effect which did not appear to be formulation-related. However, the Cmax of nadolol is only slightly sensitive to absorption rate and the relatively large variability of Cmax reduces its effectiveness as a bioequivalence metric. Findings suggest that bioequivalence criteria for highly variable drugs should be reconsidered.

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Blood samples from 101 individuals of Sadhu Chetty community domiciled in and around Madras (capital City of Tamil Nadu State, South India) were examined for HLA-A, B, C, DR and DQ antigen profiles. Phenotype, gene and haplotype frequencies were calculated and compared with the literature. Increased frequencies of HLA-B16 antigen and of the haplotype A2, B16 were characteristic. This study indicates the distinctiveness of the Sadhu Chetty population and highlights the importance of determining HLA frequencies in endogamous groups of India.

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HLA - A, B, C, DR, DQ antigen profile of South Indian Tamil-speaking Hindus of Dravidian descent was studied. Phenotype, gene and haplotype frequencies were calculated and compared with the literature. There was a complete lack of A23, A25 and A32 antigens in the sample presently monitored. Except for minor differences (higher incidence of Cw6 and DR10 antigens), the Dravidian Hindus show similarity to North Indio-Aryan and other Hindu samples. The haplotypes A1, B17; A2, B5; A2, B51; A1, DR7; B12, DR7; B13, DR2; B17, DR7; DR2, DQ1; DR3, DQ2; DR4, DQ3; DR5, DQ3; DR7, DQ2; DR11, DQ3; show significant positive linkage disequilibrium whereas A1, DR2; DR2, DQ2; DR7, DQ1 were significant for negative linkage disequilibrium in the Dravidian Hindus.

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Blood and serum samples from random individuals of three populations in south India, the first being an endogamous group from the Nilgiri hills (Tamil Nadu), the second from the Shevroy hills (Tamil Nadu), and the third from a semi-urban area of Tamil Nadu, were screened for ESD, GLO1 and Hp polymorphisms. The allelic frequencies for these markers have been estimated. High GLO1*1 (0.379) frequency was observed in the tribal Malayalis, in contrast with other Indian population groups.

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Fifty juvenile insulin dependent diabetes mellitus (JIDDM) patients of Tamil Nadu (South India) were typed for HLA-A, -B, -C, -DR, and -DQ, ESD, GLOI, C3 and HP polymorphisms. The frequencies of B8, DR3, DR4, DR53 and DQ2 antigens of the HLA system were significantly higher in the patients than in controls (relative risk, RR = 4.81; 5.14; 3.98; 3.36 and 2.53, respectively). However HLA-DR2, -DR5 and -DQ1, observed less frequently in the patient group, appear to play a role of protection against the disease (RR = 0.32; 0.30 and 0.20 respectively). HLA haplotype analysis demonstrated very high relative risk associated with two hitherto unreported haplotypes namely A3,DR1 and Cw3,DR4 (RR = 27.30 and 20.00, respectively) and also scanty distribution of the haplotypes A1,B17 and DR2,DQ1 (RR = 0.39 and 0.36, respectively) in the patient group. Among other genetic markers tested, GLOI is informative with its phenotype GLOI 2-1 showing positive association with JIDDM (RR = 4.06).

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