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Video of migration of MDA+ cells in collagen

MDA- cells migrating in collagen tracks

Video of migration of MDA- cells in collagen

Supplemental data and methods

MDA+ cells migrating in collagen tracks

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering.Significance:This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

Metabolic reprogramming is a hallmark of cancer metastasis in which cancer cells manipulate their metabolic profile to meet the dynamic energetic requirements of the tumor microenvironment. Though cancer cell proliferation and migration through the extracellular matrix are key steps of cancer progression, they are not necessarily fueled by the same metabolites and energy production pathways. The two main metabolic pathways cancer cells use to derive energy from glucose, glycolysis and oxidative phosphorylation, are preferentially and plastically utilized by cancer cells depending on both their intrinsic metabolic properties and their surrounding environment. Mechanical factors in the microenvironment, such as collagen density, pore size, and alignment, and biochemical factors, such as oxygen and glucose availability, have been shown to influence both cell migration and glucose metabolism. As cancer cells have been identified as preferentially utilizing glycolysis or oxidative phosphorylation based on heterogeneous intrinsic or extrinsic factors, the relationship between cancer cell metabolism and metastatic potential is of recent interest. Here, we review current in vitro and in vivo findings in the context of cancer cell metabolism during migration and metastasis and extrapolate potential clinical applications of this work that could aid in diagnosing and tracking cancer progression in vivo by monitoring metabolism. We also review current progress in the development of a variety of metabolically targeted anti-metastatic drugs, both in clinical trials and approved for distribution, and highlight potential routes for incorporating our recent understanding of metabolic plasticity into therapeutic directions. By further understanding cancer cell energy production pathways and metabolic plasticity, more effective and successful clinical imaging and therapeutics can be developed to diagnose, target, and inhibit metastasis.

Cancer cell migration is highly heterogeneous, and the migratory capability of cancer cells is thought to be an indicator of metastatic potential. It is becoming clear that a cancer cell does not have to be inherently migratory to metastasize, with weakly migratory cancer cells often found to be highly metastatic. However, the mechanism through which weakly migratory cells escape from the primary tumor remains unclear. Here, utilizing phenotypically sorted highly and weakly migratory breast cancer cells, we demonstrate that weakly migratory metastatic cells disseminate from the primary tumor via communication with stromal cells. While highly migratory cells are capable of single cell migration, weakly migratory cells rely on cell-cell signaling with fibroblasts to escape the primary tumor. Weakly migratory cells release microvesicles rich in tissue transglutaminase 2 (Tg2) which activate fibroblasts and lead weakly migratory cancer cell migration in vitro. These microvesicles also induce tumor stiffening and fibroblast activation in vivo and enhance the metastasis of weakly migratory cells. Our results identify microvesicles and Tg2 as potential therapeutic targets for metastasis and reveal a novel aspect of the metastatic cascade in which weakly migratory cells release microvesicles which activate fibroblasts to enhance cancer cell dissemination.

Cancer cell migration is highly heterogeneous, and the migratory capability of cancer cells is thought to be an indicator of metastatic potential. It is becoming clear that a cancer cell does not have to be inherently migratory to metastasize, with weakly migratory cancer cells often found to be highly metastatic. However, the mechanism through which weakly migratory cells escape from the primary tumor remains unclear. Here, utilizing phenotypically sorted highly and weakly migratory human breast cancer cells, we demonstrate that weakly migratory metastatic cells disseminate from the primary tumor via communication with stromal cells. While highly migratory cells are capable of single cell migration, weakly migratory cells rely on cell-cell signaling with fibroblasts to escape the primary tumor. Weakly migratory cells release microvesicles rich in tissue transglutaminase 2 (Tg2) which activate murine fibroblasts and lead weakly migratory cancer cell migration in vitro. These microvesicles also induce tumor stiffening and fibroblast activation in vivo and enhance the metastasis of weakly migratory cells. Our results identify microvesicles and Tg2 as potential therapeutic targets for metastasis and reveal a novel aspect of the metastatic cascade in which weakly migratory cells release microvesicles which activate fibroblasts to enhance cancer cell dissemination.

Glioblastoma is an aggressive brain cancer characterized by diffuse infiltration. Infiltrated glioma cells persist in the brain post-resection where they interact with glial cells and experience interstitial fluid flow. We use patient-derived glioma stem cells and human glial cells (i.e., astrocytes and microglia) to create a four-component 3D model of this environment informed by resected patient tumors. We examine metrics for invasion, proliferation, and putative stemness in the context of glial cells, fluid forces, and chemotherapies. While the responses are heterogeneous across seven patient-derived lines, interstitial flow significantly increases glioma cell proliferation and stemness while glial cells affect invasion and stemness, potentially related to CCL2 expression and differential activation. In a screen of six drugs, we find in vitro expression of putative stemness marker CD71, but not viability at drug IC

Microvesicles are small, membrane-bound vesicles that are shed from the plasma membrane of cells into the extracellular space. Microvesicles contain a variety of cargo not typically thought to be released from cells, including receptor tyrosine kinases, cytosolic signaling proteins, and microRNAs, which are transferred from donor cells to recipient cells. The transfer of microvesicle cargo can result in the transformation of recipient cells thereby supporting disease progression, including modified fibroblast metabolism, epithelial cell contractility, vascular remodeling, and immune cell inflammatory signaling. Additionally, microvesicles are believed to play prominent roles in cell-cell communication and disease progression as they are detected at elevated concentrations in diseased tissues. As microvesicle uptake by recipient cells can modulate cell function to promote disease progression, understanding the mechanisms and mechanosensitivity of microvesicle release, internalization, and the resulting signaling is crucial to fully comprehend their functions in disease. Here, we review recent advances in the understanding of actomyosin-regulated microvesicle biogenesis, microvesicle uptake via pinocytosis, and the resulting cellular transformation. We discuss the effects of altered cell contractility, mode of cell migration, and extracellular matrix compliance on microvesicle signaling, with direct implications in disease progression and identifying future therapeutic targets.

Metabolic reprogramming is a hallmark of cancer metastasis in which cancer cells manipulate their metabolic profile to meet the dynamic energetic requirements of the tumor microenvironment. Though cancer cell proliferation and migration through the extracellular matrix are key steps of cancer progression, they are not necessarily fueled by the same metabolites and energy production pathways. The two main metabolic pathways cancer cells use to derive energy from glucose, glycolysis and oxidative phosphorylation, are preferentially and plastically utilized by cancer cells depending on both their intrinsic metabolic properties and their surrounding environment. Mechanical factors in the microenvironment, such as collagen density, pore size, and alignment, and biochemical factors, such as oxygen and glucose availability, have been shown to influence both cell migration and glucose metabolism. As cancer cells have been identified as preferentially utilizing glycolysis or oxidative phosphorylation based on heterogeneous intrinsic or extrinsic factors, the relationship between cancer cell metabolism and metastatic potential is of recent interest. Here, we review current in vitro and in vivo findings in the context of cancer cell metabolism during migration and metastasis and extrapolate potential clinical applications of this work that could aid in diagnosing and tracking cancer progression in vivo by monitoring metabolism. We also review current progress in the development of a variety of metabolically targeted anti-metastatic drugs, both in clinical trials and approved for distribution, and highlight potential routes for incorporating our recent understanding of metabolic plasticity into therapeutic directions. By further understanding cancer cell energy production pathways and metabolic plasticity, more effective and successful clinical imaging and therapeutics can be developed to diagnose, target, and inhibit metastasis.

Glioblastoma is an aggressive brain cancer characterized by diffuse infiltration. Infiltrated glioma cells persist in the brain post-resection where they interact with glial cells and experience interstitial fluid flow. We recreate this infiltrative microenvironment in vitro based on resected patient tumors and examine malignancy metrics (invasion, proliferation, and stemness) in the context of cellular and biophysical factors and therapies. Our 3D tissue-engineered model comprises patient-derived glioma stem cells, human astrocytes and microglia, and interstitial fluid flow. We found flow contributes to all outcomes across seven patient-derived lines, and glial effects are driven by CCL2 and differential glial activation. We conducted a six-drug screen using four outcomes and find expression of putative stemness marker CD71, opposed to viability IC50, significantly predicts murine xenograft survival. Our results dispute the paradigm of viability as predictive of drug efficacy. We posit this patient-centric, infiltrative tumor model is a novel advance towards translational personalized medicine.

OBJECTIVE: Oxygen is essential to wound healing; therefore, accurate monitoring can guide clinical decisions. Clinical wound assessment is often subjective, and tools to monitor wound oxygen are typically expensive, indirect, and highly variable. This study demonstrates the utility of a novel, low-cost oxygen-sensing thin film for serial assessment of wound oxygenation. DESIGN: Dual-layer films were fabricated with boron oxygen-sensing nanoparticles (BNPs) impregnated into a chitosan-polycaprolactone (CS-PCL) layer for direct wound bed contact with a relatively oxygen impermeable calcium alginate surface layer. The BNPs are a dual emissive difluoroboron β-diketonate dye incorporated into poly(lactic acid) nanoparticles (BF(2)nbm(I)PLA). Under UV excitation, the BNPs emit fluorescence based on concentration and oxygen sensitive phosphorescence. The fluorescence/phosphorescence (F/P) ratio is directly proportional to oxygen concentration. METHODS: A series of in vitro oxygen challenges and in vivo murine and porcine wound healing models were used to validate the utility of the film in sensing wound oxygenation. MAIN RESULTS: In vitro testing demonstrated the oxygen-sensing capability of the BNP film and ability to shield ambient oxygen to isolate wound oxygen. In vivo testing demonstrated the ability of the film to accurately monitor relative oxygen changes in a murine wound over time, measuring a 22% F/P increase during acute healing. CONCLUSIONS: This study presents a low-cost, noninvasive, direct, and serial oxygen mapping technology to detect spatial differences in wound oxygenation. Clinical use of the films has the potential to monitor wound healing trajectories and guide wound care decisions.

Microvesicles are small, membrane-bound vesicles that are shed from the plasma membrane of cells into the extracellular space. Microvesicles contain a variety of cargo not typically thought to be released from cells, including receptor tyrosine kinases, cytosolic signaling proteins, and microRNAs, which are transferred from donor cells to recipient cells. The transfer of microvesicle cargo can result in the transformation of recipient cells thereby supporting disease progression, including modified fibroblast metabolism, epithelial cell contractility, vascular remodeling, and immune cell inflammatory signaling. Additionally, microvesicles are believed to play prominent roles in cell-cell communication and disease progression as they are detected at elevated concentrations in diseased tissues. As microvesicle uptake by recipient cells can modulate cell function to promote disease progression, understanding the mechanisms and mechanosensitivity of microvesicle release, internalization, and the resulting signaling is crucial to fully comprehend their functions in disease. Here, we review recent advances in the understanding of actomyosin-regulated microvesicle biogenesis, microvesicle uptake via pinocytosis, and the resulting cellular transformation. We discuss the effects of altered cell contractility, mode of cell migration, and extracellular matrix compliance on microvesicle signaling, with direct implications in disease progression and identifying future therapeutic targets.

Intimal stiffening upregulates endothelial cell contractility disrupting barrier integrity; however, intimal stiffening is non-uniform. The impact of local changes in intimal stiffness on proximal and distal cell-cell interactions is unknown. To investigate the range at which matrix stiffness heterogeneities impact neighboring endothelial cells within a monolayer, we built a micropillar system with adjacent regions of stiff and compliant matrix. The stiffness interface results in an oscillatory pattern of neutrophil transendothelial migration, symmetrical about the interface and well-fit by a sinusoid function. ‘Peaks’ of the sinusoid were found to have increased cellular contractility and decreased barrier function relative to ‘troughs’ of the sinusoid. Pharmacological modulation of contractility was observed to break symmetry, altering the amplitude and wavelength of the sinusoid, indicating that contractility may regulate this effect. This work illuminates a novel biophysical phenomenon of the role of stiffness-mediated cell-matrix interactions on cell-cell interactions at a distance. Additionally, it provides insight into the range at which intimal matrix stiffness heterogeneities will impact endothelial barrier function and potentially contribute to atherogenesis.

Communication between cancer cells enables cancer progression and metastasis. While cell–cell communication in cancer has primarily been examined through chemical mechanisms, recent evidence suggests that mechanical communication through cell–cell junctions and cell–ECM linkages is also an important mediator of cancer progression. Cancer and stromal cells remodel the ECM through a variety of mechanisms, including matrix degradation, cross-linking, deposition, and physical remodeling. Cancer cells sense these mechanical environmental changes through cell–matrix adhesion complexes and subsequently alter their tension between both neighboring cells and the surrounding matrix, thereby altering the force landscape within the microenvironment. This communication not only allows cancer cells to communicate with each other, but allows stromal cells to communicate with cancer cells through matrix remodeling. Here, we review the mechanisms of intercellular force transmission, the subsequent matrix remodeling, and the implications of this mechanical communication on cancer progression.

Extracellular vesicles released by cancer cells have recently been implicated in the differentiation of stromal cells to their activated, cancer-supporting states. Microvesicles, a subset of extracellular vesicles released from the plasma membrane of cancer cells, contain biologically active cargo, including DNA, mRNA, and miRNA, which are transferred to recipient cells and induce a phenotypic change in behavior. While it is known that microvesicles can alter recipient cell phenotype, little is known about how the physical properties of the tumor microenvironment affect fibroblast response to microvesicles. Here, we utilized cancer cell-derived microvesicles and synthetic substrates designed to mimic the stiffness of the tumor and tumor stroma to investigate the effects of microvesicles on fibroblast phenotype as a function of the mechanical properties of the microenvironment. We show that microvesicles released by highly malignant breast cancer cells cause an increase in fibroblast spreading, α-smooth muscle actin expression, proliferation, cell-generated traction force, and collagen gel compaction. Notably, our data indicate that these phenotypic changes occur only on stiff matrices mimicking the stiffness of the tumor periphery and are dependent on the cell type from which the microvesicles are shed. Overall, these results show that the effects of cancer cell-derived microvesicles on fibroblast activation are regulated by the physical properties of the microenvironment, and these data suggest that microvesicles may have a more robust effect on fibroblasts located at the tumor periphery to influence cancer progression.

TGF-beta plays a crucial role in tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGF-beta signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a pro-tumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response and angiogenesis. Using MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGF-beta signaling on myeloid cells (PyMT/TGFbRII-LysM) affects ECM formation in tumor tissue, specifically increases collagen and decreases fibronectin deposition, and these changes associate with mitigated tumor growth and reduced metastases. Using multiplexed five-color immunofluorescent staining and spatial analysis on a single-cell level, we discovered that reduced TGF-beta signaling on fibroblasts associates with their proximity to CD73 positive myeloid cells in tumor tissue. Consistent with these findings, in vitro gel contraction assay and Western blotting for Collagen I and pSMAD proteins confirmed that adenosine significantly downregulates TGF-beta signaling on fibroblasts. Using in vitro pharmacological approach, we found that this effect is regulated by A2a and A2b adenosine receptors. TCGA data base analysis revealed that patients with triple negative breast cancer and basal type have similar signature of adenosine and ECM profiles: high expression of A2b adenosine receptors correlates with decreased expression of Col1 and is associates with poor outcome. Taken together, our studies reveal a new role for TGF-beta signaling on myeloid cells in tumorigenesis. Discovered crosstalk between TGF-beta/CD73 on myeloid cells and TGF-beta signaling on fibroblasts can contribute to ECM remodeling and pro-tumorigenic actions of CAFs. Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Samantha Schwager, Harold Moses, Timothy Blackwell, Igor Feoktistov, Sergey Novitskiy. TGF-beta signaling on myeloid cells regulates ECM deposition in mammary carcinoma via adenosine dependent mechanisms [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1714A.

Vascular endothelial growth factor (VEGF) can mediate endothelial cell migration, proliferation, and angiogenesis. During cancer progression, VEGF production is often increased to stimulate the growth of new blood vessels to supply growing tumors with the additional oxygen and nutrients they require. Extracellular matrix stiffening also occurs during tumor progression, however, the crosstalk between tumor mechanics and VEGF signaling remains poorly understood. Here, we show that matrix stiffness heightens downstream endothelial cell response to VEGF by altering VEGF receptor-2 (VEGFR-2) internalization, and this effect is influenced by cell confluency. In sub-confluent endothelial monolayers, VEGFR-2 levels, but not VEGFR-2 phosphorylation, are influenced by matrix rigidity. Interestingly, more compliant matrices correlated with increased expression and clustering of VEGFR-2; however, stiffer matrices induced increased VEGFR-2 internalization. These effects are most likely due to actin-mediated contractility, as inhibiting ROCK on stiff substrates increased VEGFR-2 clustering and decreased internalization. Additionally, increasing matrix stiffness elevates ERK 1/2 phosphorylation, resulting in increased cell proliferation. Moreover, cells on stiff matrices generate more actin stress fibers than on compliant substrates, and the addition of VEGF stimulates an increase in fiber formation regardless of stiffness. In contrast, once endothelial cells reached confluency, stiffness-enhanced VEGF signaling was no longer observed. Together, these data show a complex effect of VEGF and matrix mechanics on VEGF-induced signaling, receptor dynamics, and cell proliferation that is mediated by cell confluency.

Objective: To determine the prevalence of post-traumatic stress disorders (PTSD) in a selected population of paediatric patients and their parents. Methodology: Twenty-three high-risk patients (serious accidents, newly diagnosed cancer) and 11 low-risk patients (simple planned surgery) and their parents were consecutively selected and assessed 6–8 weeks after the event with a DSM-IV based diagnostic interview and the PTSD Symptom Scale. Results: There is a high prevalence of PTSD in children and parents of the high-risk group. In the low-risk group such disorders are almost nonexistent. Parents in both groups report more PTSD symptoms than their children. Sex and age of the child and duration of hospitalisation have no significant influence on the development of PTSD. Conclusions: The high prevalence of PTSD in high-risk paediatric patients and their parents offers support for the applicability of a post-traumatic stress model for understanding the psychological impact of accidents, severe illnesses and their medical treatment.

The aim of this study was to obtain data concerning the incidence, reporting and follow-up of occupational exposure to blood or other body fluids (OEB). A questionnaire was distributed to employees and medical students (N=787) and official reports of OEB during the year 2003 (N=203) and their consequent follow-up (N=100) were evaluated. The percentages of needlestick injuries were 29.5% for students and 22.5% for employees. Incidence rates per 1000 employee days were 0.61 for needlestick injuries or sharp object injuries and 0.27 for mucocutaneous exposure to body fluids. The mean rate of underreporting was approximately 45%. Contrary to expectations, only 4.3% of nurses and 3.9% of doctors officially reported an OEB in 2003. The number of persons who did not attend for a serological test increased during the follow-up period. Considering all documented test results, 35 out of 100 affected persons were lost to follow-up due to default of appearance. As a consequence, the employer should provide safety devices and enforce didactical interventions with practical training and incident reporting. Periodical occupational health medicals, including serological testing, should be mandatory for all employees, including medical students and student nurses. To increase compliance after OEB, a short follow-up period using improved laboratory tests requires further discussion.

The measurement of glycated haemoglobin (HbA(1c)) is a practical and more sensitive tool than fasting plasma glucose (FPG) in screening type 2 diabetes in current practice. Its use has been limited so far by the variability of the analytical methods. The standardization process is going on, and many laboratories are currently using valid methods. Our study is consistent with the results of other groups who recommended this measurement to identify undiagnosed diabetic patients, that are about 25% to 30% in the French population. The demonstration was provided through a survey including a screening step by both HbA(1c) and G0, and a second exam with a 2 hr OGTT in a sample of positive screenees according to at least one criterion (HbA(1c)=6% or G0=1.26 g/L), as well as in a sample of negative screenees. We showed that nine confirmed diabetic subjects out of ten had HbA(1c)=6% at the screening step, while only a half had G0=1.26 g/L. Conversely, 22% of the positive screenees according to HbA(1c) were not confirmed as diabetic by the OGTT, including however more than half with abnormal glucose values. A chart for practical use is proposed to define patients at risk, the process of screening, and the patient follow-up according to the results of the tests.

We have determined the nucleotide sequence of 129,524 bases of yeast (Saccharomyces cerevisiae) chromosome XV. Sequence analysis revealed the presence of 59 non-overlapping open reading frames (ORFs) of length300 bp, three tRNA genes, four delta elements and one Ty-element. Among the 21 previously known yeast genes (36% of all ORFs in this fragment) were nucleoporin (NUP1), ras protein (RAS1), RNA polymerase III (RPC1) and elongation factor 2 (EF2). Further, 31 ORFs (53% of the total) were found to be homologous to known protein or DNA sequences, or sequence patterns. For seven ORFs (11% of the total) no homology was found. Among the most interesting protein identification in this DNA fragment are an inositol polyphosphatase, the second gene of this type found in yeast (homologous to the human OCRL gene involved in Lowe's syndrome), a new ADP ribosylation factor of the arf6 subfamily, the first protein containing three C2 domains, and an ORF similar to a Bacillus subtilis cell-cycle related protein. For each ORF detailed sequence analysis was carried out, with a full consideration of its biological function and pointing out key regions of interest for further functional analysis.

To review cases of alcoholic ketoacidosis in order to better ascertain therapeutic management.The medical files of 32 alcoholic patients with ketoacidosis hospitalized in the Saint-Pierre general hospital of the Reunion island from January 1, 1991 through 31 August 1994 were analyzed.There were 18 women and 14 men, mean age 47 years. The first clinical signs were predominated by digestive (n = 22) or neurological disorders (n = 10). Acidosis was severe (mean pH = 7.12) and always associated with a wide anion gap (mean anion gap = 35). There were 3 types of glycemic status: hypoglycemia 10 cases, normal or subnormal glycemia in 19 cases (mean glycemia = 9.3 mmol/l) and hyperglycemia above 20 mmol/l in 3 cases. Hypophosphatemia, elevated serum lactate levels and cytolytic hepatitis were the main abnormalities associated.Short-term outcome was favorable in all cases after rehydration. The use of insulin may be dangerous and needs to be avoided.

Recently available thermostable DNA polymerases result in enhanced resolution and accuracy compared to thermal enzymes used previously in fluorescent cycle sequencing. These new enzymes produce less variations in peak intensities, enhance gel resolution and are less sensitive to unspecific termination caused by either DNA structure or impurities in the DNA preparation. Optimization of nucleotide ratios and the usage of high concentrations of detergents in the sequencing reaction result in sequence readings up to 1000 bases and improve overall reliability of the sequencing protocol; this works successfully in about 90% of cases.

e21171 Background: The RAS is known for regulation of blood pressure. However, an activated RAS also supports tumor growth, in particular by its effector peptide Ang II. Elevated Ang II levels were...

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with numerous key functions in cell metabolism and growth. The human CKII has a tetrameric structure; two catalytic subunits (alpha and alpha') form the holoenzyme together with two presumably regulatory subunits (beta). The gene encoding CKII subunit beta was isolated from human genomic DNA and analyzed for its primary structure using exclusively nonradioactive procedures. The gene was found to span 4.2 kilobase pairs and to be composed of seven exons. Exon sizes range from 76 (exon 5) to 329 base pairs (bp) (exon 1), intron sizes from 145 (intron V) to 965 bp (intron II). All exon-intron junctional sequences conform to the canonical GT-AG rule. Primer extension analysis determined three transcription initiation sites, at 951, 919, and (minor) 840 bp upstream of the translation start site. The translation start is located early in the second exon; exon 1 is untranslated. The 3'-cleavage/polyadenylation signal sequence (AA-TAAA) is in the last exon at position 4173 bp relative to the first transcription initiation site. The coding sequence for CKII beta comprises 648 nucleotides identical to the published CKII beta-cDNA sequence (Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233). The upstream promoter region of the CKII beta gene contains multiple potential gene regulatory sequence elements, noticeable DNA structures, and the characteristics of a housekeeping gene (more than one transcription initiation site, lack of a TATA-box, presence of a CpG island, occurrence of multiple GC boxes and of nonstandard positioned CCAAT boxes). The CKII beta gene promoter shares common features with that of mammalian protein kinases and is closely related to the regulatory subunit gene promoter of cAMP-dependent protein kinase.

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.

Introduction Igeneon is currently testing a humanized monoclonal antibody specific for Lewis Y (IGN311) in clinical trials of passive cancer immunotherapy. The aim of the current study is to enhance the effector function of this antibody by genetically modifying the antibody producing cell line to express the glycosyl transferase GnTIII. The presence of this enzyme leads to glycosylation of the antibody with a bi-secting N-acetyl-glucosamine group and the absence of core fucosylation.

Les auteurs presentent un cas de kyste epidermoide, de situation inhabituelle (intra-sellaire) et de symptomatologie peu courante (troubles endocriniens sans manifestation ophtalmologique). Le traitement fut essentiellement chirurgical et l'evolution de la patiente est satisfaisante.

The chicken erythrocyte anion transport protein (band 3 of the erythrocyte cytoskeleton) is a central component taking part in two widely divergent functions of erythroid cells; it is a primary determinant of cytoskeletal architecture and responsible for electroneutral Cl-/HCO3- exchange across the plasma membrane. To analyze interesting aspects of the developmental regulation of this gene, we have cloned the cDNA and genomic counterparts of the erythroid-specific anion transport protein. We show that a single genetic locus for band 3 encodes two different erythroid cell-specific mRNAs, with different translational initiation sites, which predict polypeptides of sizes very close to those observed in vivo. In vitro translation and immune precipitation of synthetic mRNA derived from one putative fully encoding cDNA clone demonstrate that this clone gives rise to a protein which is identical in size and antigenicity to bona fide chicken erythroid band 3.

IN the late 1920's, the increasing demand for fast-clearing circuit breakers brought forth the development of various types of modern arc-rupturing devices. Among others, there was developed the pulsion-chamber arc-rupturing device1 which now is in satisfactory operation in thousands of breakers. The theory of expulsion action and the performance of the expulsion-chamber breakers have been reported on frequently. Test on a 115-kv system showed arcing times of approximately 1 1/2 cycles at short-circuit capacities ranging up to 1,650,000 kva.1 Tests on a 230-kv system showed arcing times of approximately 2.5 cycles at ratings up to 1,125,000 kva.2 In the middle 1930's the practice of high-speed reclosing was introduced and is now widely accepted. Interrupting capacity tests performed on a 69-kv system showed that the expulsion-chamber arc-rupturing devices gave excellent performance in conjunction with high-speed-reclosing operation.3 Following the demand for still shorter opening time, more recently five-cycle breakers have come into wide use. On highest voltages, particularly in conjunction with switching of long lines, the use of three-cycle breakers frequently is indicated.

For the first time, practical use is made of the high insulating and current interrupting ability of a high vacuum. Tests show that a vacuum switch with but 1/10-inch contact travel can clear 79-amperes at 66-kv rms has a breakdown strength of 100-kv rms.