Biomedical subjects

Stephanie Schulz

Publications and source records attributed to Stephanie Schulz.

At least 19 recordsLinked to original sources

PubMed2006

A validated quantitative assay to detect occult micrometastases by reverse transcriptase-polymerase chain reaction of guanylyl cyclase C in patients with colorectal cancer.

PURPOSE: Guanylyl cyclase C (GCC), a receptor for bacterial diarrheagenic enterotoxins, may be a prognostic and predictive marker to detect occult micrometastases in patients undergoing staging for colorectal cancer. However, quantification of GCC expression in tissues by the quantitative reverse transcription-PCR (qRT-PCR) has not undergone analytic and clinicopathologic validation. EXPERIMENTAL DESIGN: A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC. RESULTS: GCC qRT-PCR exhibited reaction efficiencies >92%, coefficients of variations <5%, linearity >6 orders of magnitude, and a limit of quantification of >25 copies of GCC cRNA. This assay confirmed that GCC mRNA was overexpressed by colorectal tumors from 41 patients, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses obtained with 164 lymph nodes from patients free of cancer and 15 nodes harboring metastases established a threshold for metastatic disease of approximately 200 GCC mRNA copies/mug total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (approximately 21%) nodes free of disease by histopathology from 6 of 23 (26%) patients, suggesting the presence of occult micrometastases. CONCLUSIONS: Quantifying GCC mRNA in tissues by RT-PCR employing external calibration standards is analytically robust and reproducible, with high clinicopathologic sensitivity and specificity. This validated assay is being applied to approximately 10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.

Adult↗

PubMed2006

Beyond gastric acid reduction: proton pump inhibitors induce heme oxygenase-1 in gastric and endothelial cells.

Proton pump inhibitors (PPIs) have been demonstrated to prevent gastric mucosal injury by mechanisms independent of acid inhibition. Here we demonstrate that both omeprazole and lansoprazole protect human gastric epithelial and endothelial cells against oxidative stress. This effect was abrogated in the presence of the heme oxygenase-1 (HO-1) inhibitor ZnBG. Exposure to either PPI resulted in a strong induction of HO-1 expression on mRNA and protein level, and led to an increased activity of this enzyme. Expression of cyclooxygenase isoforms 1 and 2 remained unaffected, and COX-inhibitors did not antagonize HO-1 induction by PPIs. Our results suggest that the antioxidant defense protein HO-1 is a target of PPIs in both endothelial and gastric epithelial cells. HO-1 induction might account for the gastroprotective effects of PPIs independently of acid inhibition, especially in NSAID gastropathy. Moreover, our findings provide additional perspectives for a possible but yet unexplored use of PPIs in vasoprotection.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2006

Tumor epithelial cell matrix metalloproteinase 9 is a target for antimetastatic therapy in colorectal cancer.

BACKGROUND: The current paradigm suggests that matrix metalloproteinase 9 (MMP-9) expressed by stromal cells is a therapeutic target in human colorectal tumors which presumably regulates metastatic disease progression. Conversely, whereas cancer cells within those tumors may induce stromal cells to produce MMP-9 and may be targets for MMP-9 activity, they are not the source of MMP-9 underlying metastasis. METHODS: MMP-9 expression in matched colorectal tumors and normal adjacent mucosa from patients and human colon cancer cell lines was examined by real-time reverse transcription-PCR, laser capture microdissection, immunoelectron microscopy, and immunoblot analysis. The role of colon cancer cell MMP-9 in processes underlying metastasis was explored in vitro by examining degradation of extracellular matrix components by gelatin zymography and formation of locomotory organelles by cell spreading analysis and in vivo by quantifying hematogenous tumor cell seeding of mouse lungs. RESULTS: Primary colorectal tumors overexpress MMP-9 compared with matched normal adjacent mucosa. In contrast to the current paradigm, MMP-9 is expressed equally by cancer and stromal cells within human colon tumors. Cancer cell MMP-9 regulates metastatic behavior in vitro, including degradation of extracellular matrix components and formation of locomotory organelles. Moreover, this MMP-9 critically regulates hematogenous seeding of mouse lungs by human colon cancer cells in vivo. CONCLUSIONS: These observations reveal that MMP-9 produced by human colon cancer, rather than stromal, cells is central to processes underlying metastasis. They underscore the previously unrecognized potential of specifically targeting tumor cell MMP-9 in interventional strategies to reduce mortality from metastatic colorectal cancer.

Adult↗

PubMed2006

Bile acids induce ectopic expression of intestinal guanylyl cyclase C Through nuclear factor-kappaB and Cdx2 in human esophageal cells.

BACKGROUND & AIMS: Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids. METHODS: Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis. RESULTS: GC-C mRNA and protein were ectopically expressed in approximately 80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor kappaB (NF-kappaB). Furthermore, deoxycholate increased NF-kappaB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-kappaB subunit p50. Moreover, a dominant negative construct for NF-kappaB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter. CONCLUSIONS: Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-kappaB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C.

Adenocarcinoma↗

PubMed2006

Are energy drinks contributing to the obesity epidemic?

The consumption of energy drinks containing sucrose and caffeine is increasing worldwide. Ten healthy women aged 18 to 22 years and fasted overnight were randomly allocated to a standardised dose of sucrose either as an "energy" drink (containing sucrose and caffeine) or lemonade on the first day and then crossed over to the alternative drink on a second day. For thirty minutes before and thirty minutes after drinking oxygen consumption and carbon dioxide production were measured in the resting subject breath-by-breath by indirect calorimetry and the rates of carbohydrate and fat oxidation calculated. Energy drink consumption apparently caused increased carbohydrate oxidation (P = 0.004) and reduced lipid oxidation (P = 0.004) compared to lemonade. The longer term effects of combined caffeine and sucrose intake, particularly in sedentary individuals, on metabolism and body fatness needs further examination.

Adolescent↗

PubMed2005

The putative tumor suppressor Cdx2 is overexpressed by human colorectal adenocarcinomas.

PURPOSE: The current paradigm suggests that the homeodomain transcription factor Cdx2, which directs the development and maintenance of the intestinal epithelium, is a tumor suppressor in the colon and rectum. Although a cardinal property of tumor suppressors is their inactivation during carcinogenesis, the expression of Cdx2 in colorectal tumors has not been compared with that in normal mucosa. Here, Cdx2 expression and function was quantified in tumors and matched normal mucosa from patients with colorectal cancer. EXPERIMENTAL DESIGN: Cdx2 expression was quantified by reverse transcription-PCR, immunoblot analysis, and immunohistochemistry. Transcriptional activity was explored by quantifying expression of an endogenous downstream target of Cdx2, guanylyl cyclase C (GCC), in tissues by quantitative reverse transcription-PCR and expression of exogenous Cdx2-specific luciferase promoter constructs in epithelial cells isolated from tumors and normal mucosa. RESULTS: Most (>80%) colorectal tumors overexpressed Cdx2 mRNA and protein compared with normal mucosa, with median fold increases of 3.6 and 1.4, respectively (P<0.002). Concomitantly, immunohistochemistry revealed elevated levels of Cdx2 in nuclei of tumor cells compared with normal epithelial cells. Further, tumors exhibited increased expression of GCC compared with normal mucosa. Moreover, cells isolated from tumors overexpressed a Cdx2-specific luciferase promoter construct compared with normal mucosal cells. CONCLUSION: These observations show, for the first time, the structural and functional overexpression of Cdx2 by human colorectal tumors compared with matched normal mucosa. They suggest that loss of Cdx2 expression or transcriptional activity is an infrequent event during tumorigenesis, which does not contribute to molecular mechanisms underlying initiation and progression of most colorectal tumors.

Adenocarcinoma↗

PubMed2005

Statistical algorithm for assuring similar efficiency in standards and samples for absolute quantification by real-time reverse transcription polymerase chain reaction.

Reverse transcription (RT) followed by the polymerase chain reaction (PCR) is the method of choice for quantifying rare transcripts in biological samples. A key assumption underlying the absolute quantification of transcripts is similar amplification efficiencies of all external standards and samples. However, efficiencies can vary between individual reactions, a problem that can be magnified when quantifying transcripts of low abundance. Here, an algorithm to assure that calibration standards and samples meet the assumption of similar amplification efficiencies underlying absolute quantification is presented. Individual reaction efficiency is estimated by fitting an exponential growth model to the fluorescence data in the exponential phase of the reaction. Next, reactions of standards with outlying estimates of amplification rates are eliminated using the boxplot outlier detection rule. Then, estimates of amplification rates of outlier-free standards are employed to define exact tolerance intervals, which are used to eliminate kinetic outliers from test samples. This algorithm was employed to eliminate kinetic outliers prior to defining the baseline expression of guanylyl cyclase C mRNA, a marker for colorectal cancer, in blood of healthy volunteers. These studies demonstrate that elimination of kinetic outliers from calibration standards and test samples improves the accuracy of absolute transcript quantification by RT-PCR.

Algorithms↗

PubMed2005

Enzymes of the last steps of chlorophyll biosynthesis: modification of the substrate structure helps to understand the topology of the active centers.

Enzymes catalyzing two of the late steps of chlorophyll biosynthesis are NADPH:protochlorophyllide oxidoreductase (POR), responsible for the light-dependent reduction of protochlorophyllide to chlorophyllide, and chlorophyll synthase that catalyses the esterification of chlorophyllide to chlorophyll. Inhibitors of these enzymes are of interest as potential herbicides. Both enzymes presumably form a complex, and the question arose whether chlorophyll synthase can react with chlorophyllide while it is still bound to POR. Here, we describe the chemical modification of protochlorophyllides and chlorophyllides with space-filling substituents at rings A, B, and E of the tetrapyrrole macrocycle and the reactivity of the modified substrates. Both enzymes tolerate the large and flexible phenylamino substituent at ring B, indicating that ring B points toward the enzyme surface while the substrate is bound. On the basis of the standard compound zinc protopheophorbide a (100% activity), the 7(1)-phenylamino derivative shows a comparable activity (83%) with POR that is higher than that of the parent formyl derivative zinc protopheophorbide b (58% activity). In contrast, the 3(1)-phenylamino derivative is less active (12%) than the parent formyl compound zinc protopheophorbide d (49% activity), indicating that the binding pocket leaves less space around ring A than around ring B. Almost no space must be left around ring E because substitution of the 13(2)-carboxymethyl ester (100% activity) by the 13(2)-carboxyethyl ester reduces the activity to 0.2%. Chlorophyll synthase leaves somewhat more space around ring E on the A side of the tetrapyrrole in the binding pocket; substitution of the 13(2)-proton (100% activity) by a methoxy group (53% activity) and an ethoxy group (11% activity) is tolerated to a certain extent, while the carbomethoxy group in this position is not accepted. Opening of ring E to a chlorin e6 dimethylester is tolerated (39% activity), while the large benzylamide residue at this site leads to the loss of activity. We conclude that the tetrapyrroles bind to both enzymes in the same direction: rings C, D, and E are oriented to the interior of the binding cleft, and rings A and B are oriented to the surface of the enzyme; this excludes simultaneous binding to both enzymes.

Bacterial Proteins↗

PubMed2005

Proliferative signaling by store-operated calcium channels opposes colon cancer cell cytostasis induced by bacterial enterotoxins.

Guanylyl cyclase C and accumulation of cGMP induced by bacterial heat-stable enterotoxins (STs) promote colon cancer cell cytostasis, serving as a tumor suppressor in intestine. Conversely, capacitative calcium entry through store-operated calcium channels (SOCs) is a key signaling mechanism that promotes colon cancer cell proliferation. The present study revealed that proliferative signaling by capacitative calcium entry through SOCs opposes and is reciprocally coupled to cytostasis mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Elimination of capacitative calcium entry employing 2-aminoethoxydiphenylborate (2-APB), a selective inhibitor of SOCs, potentiated cytostasis induced by ST. Opposition of ST-induced cytostasis by capacitative calcium entry reflects reciprocal inhibition of guanylyl cyclase C signaling. Calcium entry through SOCs induced by the calcium-ATPase inhibitor thapsigargin or the receptor agonists UTP or carbachol inhibited guanylyl cyclase C-dependent cGMP accumulation. This effect was mimicked by the calcium ionophore ionomycin and blocked by 2-APB and intracellular 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a chelator of calcium. Moreover, regulation by capacitative calcium entry reflected ligand-dependent sensitization of guanylyl cyclase C to inhibition by that cation. Although basal catalytic activity was refractory, ST-stimulated guanylyl cyclase C was inhibited by calcium, which antagonized binding of magnesium to allosteric sites required for receptor-effector coupling. These observations demonstrate that reciprocal regulation of guanylyl cyclase C signaling by capacitative calcium entry through SOCs represents one limb of a coordinated mechanism balancing colon cancer cell proliferation and cytostasis. They suggest that combining guanylyl cyclase C agonists and SOC inhibitors offers a novel paradigm for cGMP-directed therapy and prevention for colorectal tumors.

Bacterial Toxins↗

PubMed2005

C-type natriuretic peptide and guanylyl cyclase B receptor.

Guanylyl cyclases (GC) are widely distributed enzymes that signal via the production of the second messenger cGMP. The particulate guanylyl cyclases share a similar topology: an extracellular ligand binding domain and intracellular regulatory kinase-homology and cyclase catalytic domains. The natriuretic peptide receptors GC-A and -B mediate the effects of a family of peptides, atrial, B- and C-type natriuretic peptide (ANP, BNP and CNP, respectively), with natriuretic, diuretic and vasorelaxant properties. ANP and BNP, through the activation of GC-A, act as endocrine hormones to regulate blood pressure and volume, and inhibit cardiac hypertrophy. CNP, on the other hand, acts in an autocrine/paracrine fashion to induce vasorelaxation and vascular remodeling, and to regulate bone growth through its cognate receptor GC-B. GC-B, like GC-A, is phosphorylated in the basal state, and undergoes both homologous and heterologous desensitization, reflected by dephosphorylation of specific sites in the kinase-homology domain. This review will examine the structure and function of GC-B, and summarize the physiological processes in which this receptor is thought to participate.

Animals↗

PubMed2005

Guanylyl cyclase C is a marker of intestinal metaplasia, dysplasia, and adenocarcinoma of the gastrointestinal tract.

Gastrointestinal (GI) tumors continue to be major causes of cancer-related mortality, in part, reflecting metastases that escape detection by histopathology. Moreover, although approximately 10% of carcinomas arise from unknown locations, these tumors frequently originate in the GI tract. Guanylyl cyclase C (GC-C) is a receptor selectively expressed by intestinal epithelial cells whose persistent expression by colorectal carcinomas and ectopic expression by adenocarcinomas of the upper GI tract suggest its use as a marker for GI malignancies. Here, expression of GC-C protein, identified by immunohistochemistry, was examined in tissues and tumors arising from the human GI tract. Guanylyl cyclase C protein was expressed by epithelial cells from the duodenum to the rectum, but not by those in normal esophagus and stomach. Expression was retained in tubular adenomas, inflammatory bowel disease, premalignant lesions, and in primary and metastatic adenocarcinomas from the colon, including metastases to lymph nodes and liver. Moreover, GC-C was ectopically expressed in all cases of dysplasia and adenocarcinomas arising from intestinal metaplasia in esophagus and stomach. Thus, GC-C appears to be an immunohistochemical marker for identifying adenocarcinomas of unknown origin, metastases in patients undergoing staging for GI adenocarcinomas, and intestinal metaplasia, dysplasia, and tumors arising therein in the upper GI tract.

Adenocarcinoma↗

PubMed2005

Guanylyl cyclase C: a molecular marker for staging and postoperative surveillance of patients with colorectal cancer.

Staging patients with colorectal cancer defines their prognosis and therapeutic management. Unfortunately, histopathology, the current standard for staging, is relatively insensitive for detecting occult micrometastases and a significant fraction of patients are understaged and, consequently, undertreated. Similarly, current approaches to postoperative surveillance of patients with colorectal cancer detect disease recurrence at a point when interventions have little impact on survival. The detection of rare cells in tissue, for accurately staging patients, and in blood, for detecting disease recurrence, could be facilitated by employing sensitive and specific markers of disease. Guanylyl cyclase C (GCC), the receptor for the diarrheagenic bacterial heat-stable enterotoxin, is expressed selectively by cells derived from intestinal mucosa, including normal intestinal cells and colorectal tumor cells, but not by extragastrointestinal tissues and tumors. The nearly uniform expression of relatively high levels by metastatic colorectal tumors suggests that GCC may be a sensitive and specific molecular marker for metastatic colorectal cancer cells. Employing GCC reverse transcriptase PCR, occult colorectal cancer micrometastases were detected in lymph nodes that escaped detection by histopathology. Moreover, marker expression correlated with the risk of disease recurrence. Similarly, GCC reverse transcriptase PCR revealed the presence of tumor cells in blood of all patients examined with metastatic colorectal cancer and, in some studies, was associated with an increased risk of disease recurrence and mortality. These observations suggest that GCC reverse transcriptase PCR is a sensitive and specific technique for identifying tumor cells in extraintestinal sites and may be useful for staging and postoperative surveillance of patients with colorectal cancer.

Animals↗

PubMed2004

Reciprocal regulation and integration of signaling by intracellular calcium and cyclic GMP.

Calcium and guanosine-3',5'-cyclic monophosphate (cGMP) are second messenger molecules that regulate opposing physiological functions, reflected in the reciprocal regulation of their intracellular concentrations, in many systems. Indeed, cGMP and Ca2+ constitute discrete points of integration between multiple cell signaling cascades in both convergent and parallel pathways. This chapter describes the molecular mechanisms regulating intracellular Ca2+ and cGMP, and their integration in specific cellular responses.

Animals↗

PubMed2004

Nitric oxide signaling: systems integration of oxygen balance in defense of cell integrity.

PURPOSE OF REVIEW: Nitric oxide has emerged as a ubiquitous signaling molecule subserving diverse pathophysiologic processes, including cardiovascular homeostasis and its decompensation in atherogenesis. Recent insights into molecular mechanisms regulating nitric oxide generation and the rich diversity of mechanisms by which it propagates signals reveal the role of this simple gas as a principle mediator of systems integration of oxygen balance. RECENT FINDINGS: The molecular lexicon by which nitric oxide propagates signals encompasses the elements of posttranslational modification of proteins by redox-based nitrosylation of transition metal centers and free thiols. Spatial and temporal precision and specificity of signal initiation, amplification, and propagation are orchestrated by dynamic assembly of supramolecular complexes coupling nitric oxide production to upstream and downstream components in specific subcellular compartments. The concept of local paracrine signaling by nitric oxide over subcellular distances for short durations has expanded to include endocrine-like effects over anatomic spatial and temporal scales. From these insights emerges a role for nitric oxide in integrating system responses controlling oxygen supply and demand to defend cell integrity in the face of ischemic challenge. In this context, nitric oxide coordinates the respiratory cycle to acquire and deliver oxygen to target tissues by regulating hemoglobin function and vascular smooth muscle contractility and matches energy supply and demand by down-regulating energy-requiring functions while shifting metabolism to optimize energy production. SUMMARY: Insights into mechanisms regulating nitric oxide production and signaling and their integration into responses mediating homeostasis place into specific relief the role of those processes in pathophysiology. Indeed, endothelial dysfunction associated with altered production of nitric oxide regulating tissue integrity contributes to the pathogenesis underlying atherogenesis. Moreover, this central role in pathophysiology identifies nitric oxide signaling as a key target for novel therapeutic interventions to minimize irreversible tissue damage associated with ischemic cardiovascular disease.

Animals↗

PubMed2003

Functional and molecular characterization of beta-adrenoceptors in the internal anal sphincter.

The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.

Adrenergic beta-Agonists↗

PubMed2003

Expansion and proliferation of skin-homing T cells in atopic dermatitis as assessed at the single cell level.

BACKGROUND: Sensitization to aeroallergens is a frequent event in patients with atopic dermatitis (AD). The role of allergen-specific T cells in the pathogenesis of AD is, however, not fully clear. A detailed immunological characterization of allergen-specific T cells able to migrate to inflamed skin might therefore be helpful in determining the relevance of allergen-specific T cells to clinical symptoms. OBJECTIVE: To establish a simple assay to simultaneously monitor the expression of skin-related homing molecules and their proliferative response to an allergen on peripheral T cells. To evaluate whether this assay is able to identify AD patients in whom exposure to an allergen induces dermatitis. METHODS: The expression of CLA, CD7 and CD4 was simultaneously measured by flow cytometry with the DNA content as a parameter of proliferation at the single cell level. House dust mite antigen (DerP) and tetanus toxoid (TT) were used as antigens. RESULTS: CD4+ CLA+ CD7-, but not CLA- T cells from patients with AD proliferated and expanded following DerP stimulation. In contrast, TT stimulation resulted in proliferation of both, CLA+ and CLA- T cells from both, atopic and healthy donors. CONCLUSIONS: Single cell analysis revealed a predominant localization of allergen-reactive T cells within the CD7- CLA+ CD4+ T cell subset in AD. Thus, this simple assay not only confirms previously published results, but also extends our knowledge on the biology of CLA and CD7 cells in AD. Ultimately, this will be helpful in classifying those patients who might benefit from allergen avoidance therapies.

Adult↗

PubMed2003

Pathological staging and therapy of oesophageal and gastric cancer.

Oesophageal and gastric cancers are a significant cause of morbidity and mortality worldwide. Despite improvements in surgical techniques, radiation and chemotherapy, the prognosis of both cancers remains poor. Immunohistochemical and experimental studies indicate that the concept of micrometastasis is applicable to oesophageal and gastric cancer. New staging approaches, including immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of various markers, have been proposed for a more accurate staging of oesophageal and gastric cancer. Preliminary results suggest that real-time RT-PCR of markers for intestinal differentiation, such as guanylyl cyclase C (GC-C), might be useful for both the detection of premalignant conditions, such as intestinal metaplasia and the detection of micrometastasis from adenocarcinoma of the upper intestinal tract. Standard curative treatment options for oesophageal cancer include surgery or chemoradiotherapy. Chemotherapy is an option for the treatment of advanced and recurrent oesophageal cancer. Standard curative treatment for gastro-oesophageal junction and gastric cancer includes surgery and adjuvant chemoradiotherapy. Chemotherapy is an option for the treatment of advanced and recurrent gastric cancer.

Algorithms↗

PubMed2002

Nitric oxide activation of soluble guanylyl cyclase reveals high and low affinity sites that mediate allosteric inhibition by calcium.

Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.

Allosteric Regulation↗