EpCAM as multi-tumour target for near-infrared fluorescence guided surgery.

BACKGROUND: Evaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM (epithelial cell adhesion molecule) is evaluated as target for FGS in combination with the novel Artemis imaging system. METHODS: The NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein. RESULTS: All tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent. CONCLUSIONS: This study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery.

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

A multi-channel stimulator and electrode array providing a rotating current whirlpool for electrical stimulation of wounds.

When electrical stimulation is used on wounds, the electrical current has difficulty penetrating areas where there is necrotic tissue. Further, for an irregularly shaped wound, current distribution is poor in some areas of the wound since conventional two-electrode delivery systems provide the greatest current in a line directly between the electrodes. A new stimulator and electrode system is described which uses three electrodes spaced around a wound to disperse current more evenly. The stimulator senses tissue impedance and then redirects current by altering its Thevenin's output impedance for each electrode; each of the three electrodes becomes the active one in sequence while the remaining are the sink electrodes. Eight subjects were examined to test the stimulator. Electrical stimulation was applied to the skin above the quadriceps muscle at currents of 15 mA in six subjects without wounds and in two subjects with wounds. The relationship between electrode position and current dispersion on the skin was examined with a two-electrode vs. a three-electrode system to set stimulation parameters for the computer. The results showed that the three-electrode system could (1) detect areas of the skin with high impedance; (2) compensate by altering the Thevenin's output impedance at each of the three electrodes to shift current to high impedance areas; (3) provide uniform current across the skin as assessed by skin current and blood flow measurements with a laser Doppler flow imager.

A Low-Light CMOS Contact Imager With an Emission Filter for Biosensing Applications.

In this paper, a fully functional low light 128 X 128 contact image sensor for cell detection in biosensing applications is presented. The imager, fabricated in 0.18 mum CMOS technology, provides low-noise operation by employing both a modified version of the active reset (AR) technique and a modified version of the active column sensor (ACS) readout method. High-sensitivity, low noise performance of the presented sensor is well-suited for fluorescence imaging. For this purpose, an emission filter was fabricated and integrated with the sensor. The filter was fabricated using PDMS and Sudan II Blue dye mix, spin-coated and deposited in a class 1000 clean room. The designed filter is suitable for excitation at wavelengths below 340 nm and emission at 450 nm and above. The fabricated imager architecture and operation are described, noise analysis is presented and measurements from a test chip are shown. Experimental results using live neurons from the pond snail, Lymnaea stagnalis, and fluorescence polystyrene micro-beads prove the functionality of the fabricated system and indicate its biocompatiblity.

Cellular basis of diabetic nephropathy: III. In vitro GLUT1 mRNA expression and risk of diabetic nephropathy in type 1 diabetic patients.

AIMS/HYPOTHESIS: Altered glucose transporter expression has been implicated in the pathogenesis of diabetic nephropathy. There is increasing evidence that genetic factors convey risk of, or protection from, diabetic nephropathy and that the behaviour of cultured skin fibroblasts from type 1 diabetic patients may reflect these genetic influences. This study aimed to compare GLUT1 mRNA expression levels in skin fibroblasts from type 1 diabetic patients with either rapid ("fast-track", n=25) or slow ("slow-track", n=25) development of diabetic nephropathy and from non-diabetic normal control subjects (controls, n=25). METHODS: Skin fibroblasts were cultured in Dulbecco's Modified Eagle's Medium with 25 mmol/l glucose for 36 h. Total RNA was isolated, and GLUT1 mRNA levels were estimated by microarray analysis and RT-PCR. RESULTS: Levels of GLUT1 mRNA expression in skin fibroblasts from "slow-track" patients were greater than those from "fast-track" patients (p=0.02), as initially detected by microarray. GLUT1 mRNA expression levels were confirmed by RT-PCR to be higher in skin fibroblasts from "slow-track" patients (4.59+/-2.04) than in those from "fast-track" patients (3.34+/-1.2, p=0.02), and were also higher than in skin fibroblasts from control subjects (3.52+/-1.66, p=0.03). There was no statistically significant difference between levels of expression in the "fast-track" patients and the control subjects. CONCLUSIONS/INTERPRETATION: This finding is consistent with the presence of cellular protection factors against diabetic nephropathy in the "slow-track" patients. These factors could be associated with the regulation of the GLUT1 pathway and may be genetically determined.

Impact of protocol-based guidelines on the management and outcome of acute upper gastrointestinal hemorrhage in a district general hospital.

A prospective audit of acute upper gastrointestinal (GI) hemorrhage was conducted between January and September 2000 at Frimley Park Hospital to determine the impact of introducing an upper GI bleeding protocol based on Rockall's initial risk scoring system. Fifty-seven patients and 52 patients were in the pre- and postprotocol phases of the study respectively. Fifty per cent (28) of the patients in the first phase and 40% (21) of the patients in the second phase belonged to the high risk group. In the preprotocol phase, endoscopy was performed in 86% (49) of cases with 60% of patients having an esophogastroduodenoscopy within 24 h. Thirty-three per cent of the high risk group failed to have an endoscopic examination within 24 h. Only two of 57 patients required surgery and the mortality was 14%. In the postprotocol phase, endoscopy was performed in 79% (42) of patients and 68% (36) patients had endoscopy within 24 h. Only four of 21 patients belonging to the high risk group had their endoscopy after 24 h of the admission. Patients were better monitored and mortality was reduced to 7.5%. Reduction of mortality from upper GI hemorrhage followed the introduction of an agreed protocol based on risk scoring.

The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism.

PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex. To unravel the mechanism by which this assembly takes place, the following steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex. Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used. The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI. The latter replaces the PsaD subunit that is present in situ within the complex. In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes.

Continuous handrail support, oxygen uptake, and heart rate in women during submaximal step treadmill exercise.

Past research suggests that continuous handrail support during exercise attenuates physiologic responses to exercise and reduces aerobic benefits; however, this phenomenon has not been systematically studied in women exercising on the step treadmill. The effects of three levels of handrail support (continuous light, continuous very light, or no handrail support) on oxygen uptake and heart rate during step treadmill exercise were examined in 15 healthy women. Measures were obtained during 6 bouts of exercise, 3 bouts at 25 steps/min followed by 3 bouts at 33 steps/min. At both step rates, mean oxygen uptake was significantly reduced during continuous light and continuous very light handrail support as compared with no handrail support, and mean heart rate was significantly reduced during continuous light versus no handrail support. At 25 steps/min only, mean heart rate was significantly reduced during continuous very light versus no handrail support. Findings indicate that women who use even continuous light or continuous very light handrail support attenuate physiologic responses during step treadmill exercise, thereby reducing aerobic requirements and gaining suboptimal benefits from exercise.

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus.

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.

A 5-year follow up of mildly dyskaryotic smears, comparing colposcopy with expectant management.

The purpose of this study was to see if the National Health Service Cervical Screening Programme's guidelines were appropriate when they recommended that mildly dyskaryotic smears were repeated before referral for colposcopy. We identified all those with a mildly dyskaryotic smear, and followed them over 5 years, to compare those that had colposcopy and those that did not. In total, 269 women were followed and 35% of these only had the one mild smear. Of those whose smear returned to normal after the initial mild smear, 84% remained normal over the 5-year period. Of those whose smear did not return to normal, i.e. those that required a colposcopy, 74% returned to normal after the colposcopy.

Familial ureteral abnormalities syndrome: genomic mapping, clinical findings.

Abnormal development of the ureter during embryogenesis, when occurring in multiple family members, appears to be a genetically determined defect with autosomal dominant inheritance and high penetrance, which can lead to significant kidney damage, renal failure, and death. We have studied 48 individuals within a large kindred in which ureteral-related abnormalities (including vesicoureteral reflux, ureteropelvic junction obstruction, duplicated ureters, and medullary sponge kidney) were segregated. Family members who had not had previous diagnostic studies were evaluated for presence or absence of ureteral abnormalities and we attempted to map the locus for this familial ureteral abnormalities syndrome (FUAS). These studies identified 11 asymptomatic individuals, previously assumed to be unaffected, with minor abnormalities. When linkage analysis between the inheritance of ureteral abnormalities and six marker loci glyoxalase I (GLO- ), major histocompatibility antigens (HLA-A, B, and DR/DQ), D6S288, and factor XIII antigen (F13A1) on the short arm of chromosome 6 was performed, the lod scores significantly rejected linkage over a 77.1-cM distance. These findings are in contrast to previous data suggesting linkage between the presence of ureteral abnormalities and HLA, and indicate the possibility of genetic heterogeneity of FUAS.

Adhesion of cultured human kidney mesangial cells to native entactin: role of integrin receptors.

Entactin is an extracellular matrix glycoprotein which binds to laminin and is found in most renal basement membranes and in the glomerular mesangial matrix. In the present study, we have characterized specific integrin receptors on cultured human mesangial cells (CHMC) responsible for adhesion to native entactin. The integrin receptors alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and alpha 6 complexed with either beta 1 or beta 4 could be immune precipitated from detergent extracts of metabolically labeled CHMC. Adhesion assays with inhibitory anti integrin monoclonal antibodies (mab) demonstrated that CHMC use both alpha v beta 3 and a beta 1-containing integrin to bind surfaces coated with native entactin. Optimal binding of CHMC to native entactin required the participation of cations. Using wild type and mutant recombinant entactin fragments, the binding site for the alpha v beta 3 receptor was localized to the RGD sequence on the rod or E domain of entactin. CHMC adhesion to mutant full length recombinant entactin ligands lacking the E domain RGD sequence confirmed the presence of ligand binding site(s) for beta 1 integrin receptor(s). Differences in CHMC binding characteristics to recombinant and full length entactin compared to native bovine basement membrane entactin were observed. This suggests that tertiary molecular structure may contribute to entactin ligand binding properties. Primary amino acid residue sequences and tertiary structure of entactin may play roles in forming functional cell attachment sites in native basement membrane entactin.

Stepping as an exercise modality for improving fitness and function.

This article describes three stepping modes, summarizes research on stepping in healthy adults and patients with peripheral vascular disease, and discusses clinical implications and directions for future research. Stepping exercise has been shown to increase cardiorespiratory fitness in healthy adults and increase physical function in patients with peripheral vascular disease. Although further research is warranted, stepping exercise has potential value as a nursing intervention to correct alterations in physical mobility in selected populations. Stepping is proposed as an alternative exercise strategy; one which adds variety to exercise routines and may enhance long-term exercise adherence.

Relationships of cell proliferation and expression of integrin subunits and type I collagen in skin fibroblasts with renal lesions in patients with IDDM.

Previous studies have shown that cultured skin fibroblasts (SFs) from insulin-dependent diabetic mellitus (IDDM) patients with diabetic nephropathy (DN) exhibit both increased proliferation and Na+/H+ antiporter activity. The present study correlated the growth rate and mRNA expression of integrin subunits, extracellular matrix molecules, and transforming growth factor-beta in cultured SFs, with the biopsy determined rate of development of DN lesions ranging from slow to rapid in nine IDDM patients. These varying rates of development of DN lesions were expressed by a mesangial expansion score as estimated by the rate of change in mesangial fraction volume per year. Cultured SF proliferation by direct cell counts positively correlated with mesangial expansion score (r = 0.65; P < 0.05). Expression of cultured SF alpha3 integrin subunit mRNA levels, as well as type I collagen mRNA (P < 0.05 for both), but not transforming growth factor-beta mRNA levels (Northern blot analysis), were also positively correlated with mesangial expansion score. We postulate that these observations of correlations between activities of cultured SFs and the rate of progression of DN lesions may be predictive of the risk to develop clinical DN in IDDM, may be in part genetically regulated, and may be of pathogenetic importance.

Step treadmill exercise training and blood pressure reduction in women with mild hypertension.

Stepping as an exercise modality has gained increasing popularity. The step treadmill is an exercise machine which simulates actual stair-climbing. The purpose of this study was to determine the effectiveness of a 16-week supervised aerobic step treadmill exercise training protocol in reducing resting blood pressure in women with unmedicated mild hypertension. During the training protocol, subjects exercised during a self-selected appointment three times a week on the step treadmill, increasing from 20 minutes up to 60 minutes per session, at a target heart rate corresponding to 70-80% heart rate reserve for 16 weeks. Utilizing paired t-test analysis, mean resting systolic blood pressure decreased from 142.2 +/- 9.1 to 132.7 +/- 8.2 mm Hg (p < .01) and mean resting diastolic blood pressure decreased from 93 +/- 4.9 to 87.4 +/- 5.4 mm Hg (p < .01) during the 16-week protocol. Mean maximal oxygen uptake was significantly increased, whereas mean body weight did not change significantly over the 16-week period. Large scale trials are needed to further delineate the effectiveness of stepping as a health care intervention in adults with unmedicated mild hypertension, especially in women.

Distribution of integrin subunits in human diabetic kidneys.

Integrins are cell-surface protein receptors that participate in cell adhesion to multiple extracellular matrix ligands, and consist of alpha and beta chain heterodimers. This study examined altered integrin distribution in diabetic nephropathy by investigating 12 human diabetic kidney biopsies, which were compared with normal human kidney. Diabetic nephropathy is characterized by mesangial expansion and progressive thickening of the glomerular basement membrane. Based on morphometric studies of mesangial expansion, diabetic nephropathy was determined to be moderate or severe. Three different patterns (P) of altered intensity of integrin staining were observed. In the mesangial integrin P, the intensity of integrin subunit staining of mesangial cells (alpha 1, alpha 2, alpha 3, beta 1, alpha V, alpha V beta 5) was increased in moderate diabetic nephropathy and further increased in severe diabetic nephropathy. In the epithelial integrin P, integrin subunits localized to epithelial cells (alpha V, beta 3, alpha V beta 3, alpha V beta 5) were increased to the same extent in moderate and severe diabetic nephropathy. In the endothelial integrin P, integrin subunits localized to endothelial cells (alpha 3, alpha 5, alpha 6, beta 1) were increased in moderate diabetic nephropathy but returned to normal kidney staining intensity in severe diabetic nephropathy. From these observations, it was concluded that there is significant alteration in the expression of integrin subunits in diabetic nephropathy that is related to the severity of diabetic mesangial expansion. Additionally, the spectrum of integrin subunit alteration appears to be unique to individual glomerular cell types. Given the role of integrins in cell-surface interactions with extracellular matrix components, abnormalities in the expression of these molecules may be important in the pathogenesis of diabetic nephropathy.

Secondary desertification due to salinization of intensively irrigated lands: The Israeli experience.

Secondary salinization of intensively irrigated lands is an increasingly alarming redesertification process experienced in many irrigated regions of the developed countries. The major cause is a profound interference in the geochemical/salt balances of irrigated regions. A case-in-point is the recent salinization of the Yizre'el Valley, a 20,000 ha intensively irrigated region in Israel. The extremely intensive and advanced agroecosystem developed in the region since the 1940s included pumping and importing irrigation water by the National Water Carrier, large-scale reclamation and reuse of municipal sewage water, winter flood impoundment in reservoirs for summer irrigation, and cloud seeding to enhance rainfall. Modern irrigation methods were applied, including sprinkler, trickle, moving-line, and center-pivot systems. Water use efficiency at any level was very high. Nevertheless, large-scale salinization of regional water resources and many fields had developed in the mid-1980s. Reconstructing and evaluating the water and salt balances of the Yizre'el Valley (using Cl as the representative salt constituent) shows that as water use in the valley increased to about 60 million m(3) per year, the importing of soluble salts by water totaled 15,000 tons of Cl per year. Recirculated salt - salt picked up by impounded surface water and applied to fields - increased significantly and in the late 1980s amounted to more than 9,000 tons Cl per year. The source of recirculated salts was the accumulated salts in soils and in the shallow aquifer in the valley, which were leached by floodwater or drained or infiltrated into reservoirs, grossly and adversely affecting water quality. Analysis of the Yizre'el Valley's case points to the utmost importance of maintaining the geochemical balances in addition to increasing irrigation efficiency. An irrigated region may achieve geochemical balance by the following means: limiting the extent of irrigated areas, developing a well-maintained drainage system that drains tail-water and salinized shallow-aquifer water, and devoting a significant portion of water for regional leaching. The sustained long-term productivity of irrigated lands in arid zones crucially depends on correctly managing water and soil resources. Regional management of irrigated lands to prevent secondary desertification will be aimed at carefully balancing the undisputed benefits of irrigation with the long-term (on time scales of 10 to 100 years) detrimental processes set in motion when irrigation is introduced to arid and semiarid zone soils.

Angiotensin converting enzyme inhibitors: inhibition of growth, a novel mechanism of action.

Angiotensin II, a known vasopressor agent, recently has been implicated as a growth factor in the heart and vascular smooth muscle. Angiotensin converting enzyme (ACE) inhibitors, which block angiotensin II synthesis, have been found to prevent or reverse cellular growth in the heart and vasculature--a novel mechanism of action. Further study is required to determine the precise mechanisms through which angiotensin II promotes growth as well as the mechanisms through which ACE inhibitors block growth. Large scale clinical trials are needed to delineate further the effectiveness of ACE inhibitors in humans, especially regarding vascular smooth muscle growth.

Entactin/nidogen: synthesis by bovine corneal endothelial cells and distribution in the human cornea.

PURPOSE: The purposes of this study were to determine whether entactin/nidogen (E/N) is synthesized and secreted by corneal endothelial cells and to characterize the distribution of E/N in the human cornea. METHODS: Cultured bovine corneal endothelial cells were metabolically labeled with [35S]methionine. Newly synthesized E/N was detected in cell lysates and culture medium by immunoprecipitation, using monoclonal anti-E/N antibodies, polyacrylamide gel electrophoresis, and autoradiography. The presence of E/N in the subendothelial extracellular matrix was demonstrated by Western blot analysis of solubilized extracellular matrix proteins. The distribution of E/N in normal human corneas was studied by indirect immunofluorescent staining of frozen sections, using monospecific anti-E/N antibodies. RESULTS: E/N was detected in the basement membrane (BM)-like extracellular matrix deposited by corneal endothelial cells, as well as in cell lysates and culture medium. Immunofluorescence studies revealed the presence of E/N in both the epithelial and endothelial BM and to a much lower extent in the stroma. E/N was detected throughout the thickness of the epithelial BM, but its staining decreased in intensity toward the central part of the cornea. In the endothelial BM (Descemet's membrane), E/N fluorescence was limited to its most posterior portion, produced postnatally. CONCLUSIONS: Corneal endothelial cells synthesize and secrete E/N, E/N was found in both the epithelial and endothelial basement membranes but was primarily localized to the posterior portion of Descemet's membrane and the periphery of the epithelial BM. The authors suggest that E/N may be important in healing processes of corneal injuries and in the pathogenesis of diseases involving the postnatal region of Descemet's membrane.