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Cholangiocarcinoma (CCA), or cancer of bile duct epithelial cells, is a very aggressive malignancy characterized by early lymphangiogenesis in the tumor microenvironment (TME) and lymph node (LN) metastasis which correlate with adverse patient outcome. However, the specific roles of lymphatic endothelial cells (LECs) that promote LN metastasis remains unexplored. Here we aimed to identify the dynamic molecular crosstalk between LECs and CCA cells that activate tumor-promoting pathways and enhances lymphangiogenic mechanisms. Our studies show that inflamed LECs produced high levels of chemokine CXCL5 that signals through its receptor CXCR2 on CCA cells. The CXCR2-CXCL5 signaling axis in turn activates EMT (epithelial-mesenchymal transition) inducing MMP (matrix metalloproteinase) genes such as GLI, PTCHD, and MMP2 in CCA cells that promote CCA migration and invasion. Further, rate of mitochondrial respiration and glycolysis of CCA cells was significantly upregulated by inflamed LECs and CXCL5 activation, indicating metabolic reprogramming. CXCL5 also induced lactate production, glucose uptake, and mitoROS. CXCL5 also induced LEC tube formation and increased metabolic gene expression in LECs. In vivo studies using CCA orthotopic models confirmed several of these mechanisms. Our data points to a key finding that LECs upregulate critical tumor-promoting pathways in CCA via CXCR2-CXCL5 axis, which further augments CCA metastasis.

Tumor metastasis to the draining lymph nodes is critical in patient prognosis and is tightly regulated by molecular interactions mediated by lymphatic endothelial cells (LECs). The underlying mechanisms remain undefined in the head and neck squamous cell carcinomas (HNSCCs). Using HNSCC cells and LECs we determined the mechanisms mediating tumor-lymphatic cross talk. The effects of a pentacyclic triterpenoid, methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF 3 DODA-Me), a potent anticancer agent, were studied on cancer-lymphatic interactions. In response to inflammation, LECs induced the chemokine (C-X-C motif) ligand 9/10/11 chemokines with a concomitant increase in the chemokine (C-X-C motif) receptor 3 (CXCR3) in tumor cells. CF 3 DODA-Me showed antiproliferative effects on tumor cells, altered cellular bioenergetics, suppressed matrix metalloproteinases and chemokine receptors, and the induction of CXCL11-CXCR3 axis and phosphatidylinositol 3-kinase/AKT pathways. Tumor cell migration to LECs was inhibited by blocking CXCL11 whereas recombinant CXCL11 significantly induced tumor migration, epithelial-to-mesenchymal transition, and matrix remodeling. Immunohistochemical analysis of HNSCC tumor arrays showed enhanced expression of CXCR3 and increased lymphatic vessel infiltration. Furthermore, The Cancer Genome Atlas RNA-sequencing data from HNSCC patients also showed a positive correlation between CXCR3 expression and lymphovascular invasion. Collectively, our data suggest a novel mechanism for cross talk between the LECs and HNSCC tumors through the CXCR3-CXCL11 axis and elucidate the role of the triterpenoid CF 3 DODA-Me in abrogating several of these tumor-promoting pathways., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

This paper uses confidential Census data, specifically the 1990 and 2000 Census Long Form data, to study the income dispersion of recent cohorts of migrants to mixed-income neighborhoods. We investigate whether neighborhoods with high levels of income dispersion attract economically diverse in-migrants. If recent in-migrants to mixed-income neighborhoods exhibit high levels of income dispersion, this is consistent with stable mixed-income neighborhoods. If, however, mixed-income neighborhoods are comprised of homogenous low-income (high-income) cohorts of long-term residents combined with homogenous high-income (low-income) cohorts of recent arrivals, this is consistent with neighborhood transition. Our results indicate that neighborhoods with high levels of income dispersion do in fact attract a much more heterogeneous set of in-migrants, particularly from the tails of the income distribution. Our results also suggest that the residents of mixed-income neighborhoods may be less heterogeneous with respect to lifetime income.

This paper uses confidential Census data, specifically the 1990 and 2000 Census Long Form data, to study demographic processes in neighborhoods that gentrified during the 1990's. In contrast to previous studies, the analysis is conducted at the more refined census-tract level, with a narrower definition of gentrification and more closely matched comparison neighborhoods. Furthermore, our access to individual-level data with census tract identifiers allows us to separately identify recent in-migrants and long-term residents. Our results indicate that, on average, the demographic flows associated with the gentrification of urban neighborhoods during the 1990's are not consistent with displacement and harm to minority households. In fact, taken as a whole, our results suggest that gentrification of predominantly black neighborhoods creates neighborhoods that are attractive to middle-class black households.

There has been a dramatic increase in the availability of case studies for use in the biology classroom, and perceptions of the effectiveness of case-study-based learning are overwhelmingly positive. Here we report the results of a study in which we evaluated the ability of interrupted case studies to improve critical thinking in the context of experimental design and the conventions of data interpretation. Students were assessed using further case studies designed to evaluate their ability to recognize and articulate problematic approaches to these elements of experimentation. Our work reveals that case studies have broad utility in the classroom. In addition to demonstrating a small but statistically significant increase in the number of students capable of critically evaluating selected aspects of experimental design, we also observed increased student engagement and documented widespread misconceptions regarding the conventions of data acquisition and analysis.

Wound healing has evolved from a science of clinical observation to a study of molecular biology and cellular physiology. This article will review the medical literature to provide a basis for understanding the current science of wound healing.

2-Amino-4-azaindoles have been identified as a structurally novel class of BK(Ca) channel openers. Their synthesis from 2-chloro-3-nitropyridine is described together with their in vitro properties assessed by 86Rb(+) efflux and whole-cell patch-clamp assays using HEK293 cells stably transfected with the BK(Ca) alpha subunit. In vitro functional characterization of BK(Ca) channel opening activity was also assessed by measurement of relaxation of smooth muscle tissue strips obtained from Landrace pig bladders. The preliminary SAR data indicate the importance of steric bulk around the 2-amino substituent.

We have discovered a novel series of N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl] amides that are potent openers of K(ATP) channels and investigated structure-activity relationships (SAR) around the 1,2-disubstituted naphthyl core. A-151892, a prototype compound of this series, was found to be a potent and efficacious potassium channel opener in vitro in transfected Kir6.2/SUR2B cells and pig bladder strips. Additionally, A-151892 was found to selectively inhibit unstable bladder contractions in vivo in an obstructed rat model of myogenic bladder function

Several lines of evidence indicate that calreticulin has lectin-like properties. As a molecular chaperone, calreticulin binds preferentially to nascent glycoproteins via their immature carbohydrates; this property closely resembles that seen for calnexin, a chaperone with extensive molecular identity to calreticulin. A cell surface form of calreticulin also exhibits lectin-like properties, binding specific oligomannosides including those covalently linked to laminin. In the present study we examined the interaction between calreticulin and laminin by means of surface plasmon resonance. The results show that calreticulin specifically binds to glycosylated laminin but fails to specifically bind tunicamycin-derived unglycosylated laminin or bovine serum albumin. Calreticulin binding to glycosylated laminin requires calcium and is abolished in the presence of EDTA. Scatchard analysis of binding yields an apparent association constant, Ka, of 2.1 +/- 0.9 x 10(6) m-1 while kinetic analysis yields an estimate of the association on rate, (Kassoc), as 2 x 10(5) m-1 s-1. The composite results support calreticulin's lectin-like properties as well as its proposed role in laminin recognition, both in the cell interior and on the cell surface.

B16 mouse melanoma cells adhere to and spread on laminin. We have previously shown that cell spreading is uncoupled from adhesion when unglycosylated laminin is used as a substratum; spreading was restored by a Pronase digest of laminin which became inactive when it was specifically depleted of its mannoside peptides; spreading was also specifically restored by mannosides such as mannan, Man9, and Man6, but not Man3. The effector mannosides bind to a cell surface receptor, previously shown by direct and indirect methods. We have now identified the receptor as cell surface calreticulin by isolating it via mannan affinity chromatography and showing its sequence identity with mouse calreticulin. Anti-calreticulin antibodies confirm this identity, decorate the B16 cell surface, and block cell spreading. Purified B16 cell calreticulin from whole cell lysates successfully competes with cell surface calreticulin and prevents cell spreading. The composite data implicate cell surface calreticulin as a putative lectin that must be occupied to initiate spreading of laminin-adherent B16 cells.

An anionic glycolipid and phospholipid binding protein was characterized in detergent-solubilized rat brain synaptosomes using a synthetic, polyvalent radiolabeled ganglioside-protein conjugate as radioligand. Gangliosides are prominent cell surface glycoconjugates in vertebrate brain, where they may function in membrane protein regulation or in cell-cell recognition. The neoganglioprotein (GT1b)13BSA was radioiodinated and used to probe solubilized synaptosomal proteins for ganglioside binding activity using a receptor-ligand precipitation assay. Binding data revealed a high affinity (KD = 1 nM), saturable (Bmax = 173 pmol/mg protein) binding activity that was proteinase sensitive, calcium independent and maximal at neutral pH. Size exclusion chromatography of the synaptosomal (GT1b)13BSA binding activity indicated a M(r) of approximately 28 kDa. Binding activity with similar characteristics was solubilized from other rat tissues, with activity from sciatic nerve = muscle > synaptosomes > central nervous system myelin = liver. Gangliosides added as mixed detergent-lipid micelles inhibited (GT1b)13BSA binding: GT1b, GD1a and GD1b were the most effective inhibitors (IC50 approximately 200 nM), while GM1 and GM3 were 5-fold less effective. In addition, the sphingolipids sulfatide and sialylneolactotetraosylceramide were effective inhibitors, with IC50 values of 300 nM and 200 nM, respectively. The neutral sphingolipid GA1 did not block (GT1b)13BSA binding. Phosphatidylcholine and phosphatidylethanolamine were non-inhibitory, however phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol inhibited half-maximally at 200-300 nM. Inhibition by both gangliosides and anionic phospholipids was competitive with (GT1b)13BSA. We conclude that a binding protein for anionic glycolipids and phospholipids is distributed on nerve and muscle membranes.

We report a case of trochlear nerve palsy that was the sole initial sign of an aneurysm of the superior cerebellar artery. A craniotomy confirmed the anatomical compression of the trochlear nerve by this aneurysm. The clipping of the aneurysm resulted in the resolution of the trochlear nerve palsy.

The effects of various agents on the cleavage of serum albumin, interferon, immunoglobulin and complement component C1q by the extracellular protease from Staphylococcus aureus were analysed by SDS-polyacrylamide gel electrophoresis. Arachidonic acid moderately stimulated the proteolysis of serum albumin, interferon and complement component. Phosphatidic acid effectively enhanced the proteolysis of serum albumin and IgG, whereas it inhibited the cleavage of IgM. The proteolysis of IgG was appreciably enhanced by sphingosine. In contrast, phosphatidyl choline and phosphatidyl glycerol were shown to have an inhibitory effect on the proteolysis of IgG and IgM. Phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine also inhibited the proteolysis of IgG. The failure of any of these agents to exert a persistent effect on the cleavage of all substrates, revealed the complexity of the interactions among the agent, the substrate and the protease.

The synthesis and release of plasminogen activators (PAs) in co-cultures of embryonic rat dorsal root ganglion nerve cells (NCs) and Schwann cells (SCs) were examined by metabolic labeling, immunoprecipitation, immunodepletion, SDS-PAGE, zymography, and a two-step esterolytic assay. Metabolic labeling of SC cultures followed by immunoprecipitation of the conditioned medium (CM) demonstrated that cultured SCs synthesized and released tissue type PA (tPA). Failure of amiloride to inhibit PA activity in SCCM indicated that urokinase PA (uPA) was unlikely to contribute significantly to PA activity in SCCM. Experimental manipulation of the NCs and SCs suggested that NCs regulated SC derived PA. Total PA activity increased in SCCM 10-14-fold by 6 days after removal of NCs. Multiple molecular weight forms of PAs were detected by SDS-PAGE followed by zymography. A PA approximately 95 kDa was absent in co-cultures of SCs + NCs but prominent by 4 days postdenervation; PA approximately 50-70 kDa increased through 8 days postdenervation and PA approximately 25 kDa, present in SC + NC cultures, was absent 8 days after removal of NCs. Upon reintroduction of NCs to denervated cultures (SCs), the pattern of PAs detected in culture medium was transitional between innervated and denervated cultures. Immunodepletion experiments using conditioned medium from denervated SC cultures indicated that various molecular weight forms of PA detected in SCCM by zymography were immunologically related to tPA. These studies demonstrate that SCs synthesized and released tPA in a tissue culture model of peripheral nerve and that one mechanism for regulation of PA released by SCs was by association with NCs. This regulation occurred in cultures of both myelinating and nonmyelinating Schwann cells and thus was not dependent on the state of myelination.

Mg2(+)-chelates of several nucleoside triphosphates were shown to increase the inactivation of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) by 0.6 M guanidine hydrochloride, with ATP-Mg2+ having the greatest effect; unchelated forms did not significantly affect inactivation. Since catalytic activity has been associated with the C-terminal half of the molecule, these results were interpreted as indicating a destabilization of this C-terminal region by binding of these chelates to the substrate nucleotide sites, with the particular effectiveness of ATP-Mg2+ reflecting the specificity for this species as a phosphoryl donor. These compounds were also shown to bind to the N-terminal half of the enzyme, as judged by their ability to protect against denaturation by guanidine hydrochloride and subsequent digestion with trypsin. Both free and Mg2(+)-chelated forms afforded protection, with the unchelated nucleotides being most effective; a preference for ATP was seen only with the chelated forms. Thus, it was concluded that the N-terminal half of hexokinase contains a relatively nonspecific nucleotide binding site, distinct from the substrate nucleotide site previously shown to reside in the C-terminal half. On the basis of this same ability to protect the N-terminal half against denaturation and proteolysis, several other polyanionic ligands were shown to bind to this region of the molecule. These included inorganic phosphate, its analogs, sulfate and arsenate, and its homologs, pyrophosphate and tripolyphosphate. All of these anionic ligands were also shown to antagonize inhibition by the glucose 6-phosphate (Glc-6-P) analog, 1,5-anhydroglucitol 6-phosphate. The allosteric site for binding of Glc-6-P has previously been shown to reside in the N-terminal half of the molecule, and it is suggested that the antagonism of inhibition by Glc-6-P (or its analog) by these anionic ligands results from interaction with an anion binding site for which the 6-phosphate group of inhibitory hexose 6-phosphates must compete. A model depicting possible relationships between ligand binding sites on brain hexokinase, and how their interactions might lead to observed regulatory properties, is developed based on these and previous studies of ligand binding as well as evidence that mammalian hexokinases (Mr 100,000) have evolved by duplication and fusion of a gene coding for an ancestral hexokinase with Mr 50,000 and which, like the mammalian enzyme, was sensitive to inhibition by Glc-6-P.

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been studied by differential scanning calorimetry. In "high-ionic-strength" buffer (50 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), and assuming two-state behavior with calorimetric enthalpy equal to van't Hoff enthalpy, the endotherm could be deconvoluted into two transitions with Tm values of about 48 and 51 degrees C and enthalpies of about 109 and 112 kcal/mol, respectively. A similar endotherm was seen when glucose or glucose 6-phosphate was present, except that Tm values for both transitions were increased. The glucose analog, N-acetylglucosamine, had no observable effect on the endotherm, which is in agreement with previous studies indicating that this ligand, unlike glucose and glucose 6-phosphate, does not induce conformational changes that lead to increased stability of the enzyme. In "low-ionic-strength" buffer (5 mM Tris-Cl, 0.5 mM EDTA, 10 mM monothioglycerol, pH 8.5), the transitions were partially resolved even in the absence of ligands, with Tm values of about 49 and 55 degrees C. Due to difficulties with erratic baseline behavior under the low-ionic-strength conditions, enthalpies were not routinely determined, but these appeared to be similar to those seen in high-ionic-strength buffer. Also similar was the increase in stability, as reflected by the increase in Tm for both transitions, when glucose or glucose 6-phosphate was present. Correlation of these transitions with specific regions of the molecule was established by analysis of enzyme in which the domain corresponding to the first transition was selectively denatured by a partial scan in the calorimeter. Subsequent rescanning of these samples showed only the second transition, confirming the selective denaturation of the domain corresponding to the first transition and retention of the folded structure of the second domain. Discrimination between denatured (first transition) and undenatured (second transition) domains was based on the markedly increased susceptibility of the denatured region to tryptic digestion; regions of the molecule that retained their folded structure and resistance to proteolysis were identified by immunoblotting techniques using monoclonal antibodies recognizing epitopes having defined locations within the overall sequence. Based on this analysis, the first transition corresponds to unfolding of the C-terminal half of the molecule, with the second transition resulting from unfolding of the more stable N-terminal half. The order of unfolding could be reversed in the presence of ATP-Mg2+ and N-acetylglucosamine, conditions which have been shown to result in selective stabilization of the C-terminal domain.

Selective stabilization of either the N- or C-terminal half (by ligands binding to these regions) of rat brain hexokinase against partial denaturation with guanidine hydrochloride and subsequent digestion with trypsin has provided a means for isolating these regions, referred to as N fragment and C fragment, respectively, in quantities adequate for characterization. The N fragment (mol wt 52 kDa) is devoid of catalytic activity. In contrast, the C fragment (mol wt 51 kDa) has a specific activity of about 110 U/mg, nearly twice that (60 U/mg) of the intact 100-kDa enzyme, indicating that the kappa cat is virtually identical for both species. Unlike the parent enzyme, the C fragment is quite sensitive to inhibition by Pi (competitive vs ATP, noncompetitive vs Glc); sulfate and arsenate, but not acetate, inhibit with effectiveness similar to that seen with Pi. The Glc-6-P analog, 1,5-anhydroglucitol-6-P, also inhibits the C fragment (competitive vs ATP, uncompetitive vs Glc). Both N and C fragments bind to Affi-Gel Blue, an affinity matrix bearing a covalently attached analog of ATP, and are eluted by hexose 6-phosphates competitive with nucleotide binding to the parent enzyme. Based on the ability of various hexoses and hexose 6-phosphates (and analogs) to protect against guanidine-induced denaturation and subsequent proteolysis it is concluded that both fragments contain discrete sites for hexoses and hexose 6-phosphates, with specificities resembling those seen for the binding of these ligands to the parent enzyme. Synergistic interactions between the hexose and hexose-6-P binding sites, previously seen with the parent enzyme, are also observed with the C fragment but not the N fragment. The existence of binding sites for hexoses and hexose 6-phosphates on both halves conflicts with previous binding studies demonstrating a single hexose binding site and a single hexose 6-phosphate binding site on the intact 100-kDa enzyme, leading to the conclusion that one of each pair of sites must be latent in the intact enzyme, becoming manifest only in the isolated discrete halves. Several investigators have previously suggested that the 100-kDa mammalian hexokinases evolved by duplication and fusion of a gene encoding an ancestral 50-kDa Glc-6-P-insensitive hexokinase, similar to the present-day yeast enzyme, with sensitivity to Glc-6-P resulting from evolution of a duplicated catalytic site into a regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)

After denaturation in 0.6 M guanidine hydrochloride, rat brain hexokinase becomes highly susceptible to proteolysis by trypsin. Glucose 6-phosphate (Glc-6-P) and its analog, 1,5-anhydroglucitol 6-phosphate, selectively protect the N-terminal half of the molecule from proteolysis. These compounds do not protect the C-terminal half of the molecule, nor do they protect enzyme activity; the Glc analog, N-acetylglucosamine, does protect the C-terminal domain and catalytic activity, but does not prevent proteolysis of the N-terminal half of the molecule. These results are consistent with previous work [M. Nemat-Gorgani and J. E. Wilson (1986) Arch. Biochem. Biophys. 251, 97-103; D. M. Schirch and J. E. Wilson (1987) Arch. Biochem. Biophys. 254, 385-396] demonstrating that binding sites for both hexose and nucleotide substrates, and thus catalytic function, are associated with a 40-kDa domain located at the C-terminus of the enzyme. They further demonstrate that the binding site for the allosteric effector, Glc-6-P, lies in the N-terminal half of the molecule and is distinct from the catalytic site. Using protection against proteolysis as a reflection of binding, it is shown that the Glc-6-P binding site in the N-terminal region has all the characteristics described for the allosteric effector site on this enzyme in terms of affinity for Glc-6-P, specificity, and synergistic interactions with the hexose binding site in the C-terminal region of the molecule. This disposition of catalytic and regulatory functions in discrete halves of the molecule is consistent with suggestions by several investigators that mammalian hexokinases evolved by a process of duplication and fusion of an ancestral gene coding for a hexokinase similar to the present-day yeast enzyme, with the regulatory site of mammalian hexokinases having evolved from what was originally a catalytic site.

Synovial cysts are known to occur in patients with rheumatoid arthritis. Their presentation in the hip joint, however, continues to be a diagnostic challenge. A case is presented involving a patient with a large synovial cyst of the hip, emphasizing the need for the orthopaedic surgeon to include synovial cyst in the differential diagnosis of a painless groin mass, particularly in patients with rheumatoid arthritis.