If you are having trouble viewing this email, click on this link.
To order any of our products, please go to our website or visit one of our distributors:

Methicillin Resistant Staphylococcus aureus (MRSA) Enzyme Assays.
Methicillin-resistant Staphylococcus aureus (MRSA) is a resistant variation of the common bacterium Staphylococcus aureus that has evolved the ability to survive treatment with beta-lactam antibiotics, including penicillin, methicillin, and cephalosporins. It is also sometimes referred to as multiply-resistant Staphylococcus aureus or oxacillin-resistant Staphylococcus aureus (ORSA) or multiple resistant staph infection. Non-multi-resistant MRSA (NORSA) has been defined as susceptibility to two or more non beta-lactam antibiotics including ciprofloxacin, erythromycin, tetracycline and trimethoprim-sulfamethoxazole.The MRSA organism has been sub-categorized as community-acquired MRSA (CA-MRSA) or hospital-acquired MRSA (HA-MRSA) depending upon the circumstances of acquiring the disease. Current data indicate that these represent distinct strains of the bacterial species. MRSA has been found to be especially troublesome in hospital-acquired (nosocomial) infections where patients have open wounds, invasive devices, or weakened immune systems and can be at greater risk for infection than the general public. Estimates recently published by the Centers for Disease Control (CDC) based on US data put the number of MRSA associated fatalities at more than 18,000/yr. This places MRSA infection fatalities at levels approaching those for the AIDS epidemic in the US.

Several methods have been developed to screen for MRSA including PCR assays and agar plate assays. All of these methods utilize MRSA selective agars for selective culture of the bacteria. These include: MRSA ID® (Biomerieux), MRSASelect ® (Bio-Rad Laboratories), CHROMagar MRSA (CHROMagar, Paris,France; or the CHROMagar MRSA available from Becton Dickinson Microbiology Products US. The exact composition of these agars are proprietary, but mostly contain a selective agent in a culture agar (similar to mannitol salt agar with 8 μg/ml cefoxitin) as well as a chomogenic substrate (like X-Phos) to mark the growing colonies. Recently, Biosynth AG (Switzerland) has teamed up with Invitrogen-Molecular Probes to develop a fluorogenic assay for MRSA that utilizes the precipitating fluorescent substrate ELF-Phos which produces a bright (green) fluorescent precipitate on selective agar plates at the site of MRSA colony growth. In contrast to fluorescence in solution, solid state fluorescence is rare due to self-quenching in the solid state and most molecules that display solid state fluorescence remain fluorescent in solution as well. This, however, is not the case for ELF-Phos substrate used in these assays, providing a new, definitive assay for colony growth. For more information about these assays and substrates, please see the references below or visit our website.

• Klevens, RM, R. Morrison,MA. Joelle Nadle, J, Petit, S, Gershman, K, Ray, S, Harrison, LH, Lynfield,R, Dumyati, G, Townes,JM, Craig, AS, Zell, ER, Fosheim, GE, McDougal, LK, Carey,RB, Fridkin,SK, Invasive Methicillin Resistant Staphylococcus aureus Infections in the United States. JAMA, 2007. 298(15):1763-1771.
• Okuma K, Iwakawa K, Turnidge J, et al (2002). "Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community". J Clin Microbiol 40(11): 4289-94.
• Naleway, JJ, Fox, CMJ, Robinhold, D, Terpetsching, E, N.A. Olsen NA, Haugland, RP (1994) " Synthesis and use of new fluorogenic precipitating substrates" Tetrahedron Lett. 46: 8569–8572. 
• Haugland RP, Zhang Y, Yue ST, Terpetschnig E, Olson NA, Naleway JJ, Larison; KD, Huang Z, (1994) "Enzymatic analysis using substrates that yield fluorescent precipitates" US Patent 5,316,906

Labeling with MRI Contrast Agents.

Magnetic Resonance Imaging has become a conventional technique for monitoring and evaluating disease in vivo. The ability to track small molecules by MRI is beginning to become useful for therapeutic or diagnostic evaluations. In order to track these molecules, two classes of contrast materials have been routinely used. Both gadolinium chelates and iron oxide nanoparticles or microparticles with various surface modifications have been developed for such applications. Gadolinium chelates, such as gadopentate dimeglumine, are the most widely used paramagnetic contrast material. These chelates are effective contrast agents because of their seven unpaired electrons. Unpaired electrons produce a magnetic moment that increases relaxation constants in MRI and thus alters signal intensity in the region near the contrast material.

Iron oxide particles are a second class of superparamagnetic MRI contrast agents in use today. These particles range in size from tens of nanometers in diameter, termed ultra-small superparamagnetic iron oxide (USPIO), to 100 nm (superparamagnetic iron oxide [SPIO]), to some larger than 1 µm, known as micrometer-sized iron oxide particles (MPIOs). These compounds consist of magnetite (iron oxide) cores, which are further coated with either dextran or siloxanes and encapsulated by a polymer or further modified to facilitate internalization into cells and tissues. Such particles function as contrast agents by creating a large dipolar magnetic field gradient that is transferred to protons in close proximity to the particle. With the use of signal amplification, applications of iron oxide particles include labeling of therapeutic stem cell preparations with a small fraction of labeled cells have allowed cell tracking in vivo.

The ability to internalize these magnetic particles has been demonstrated in a variety of cell lines. The size of the particle and the rate at which it is internalized is dependent on specific transmembrane mechanisms. White blood cells, including macrophages, neutrophils and eosinophils, are capable of rapidly phagocytosing even large particles using specific receptor-mediated endocytosis. A wider range of cells have been shown to be able to internalize very small particles through pinocytosis. These include fibroblasts, immortalized rat progenitor cells, human hepatocellular liver carcinoma cells and human hematopoietic progenitor cells. To date, the majority of nonspecific cellular labeling has so far involved macrophages. Particle size as well as coating are important parameters for internalization. For example, particles coated with the monomeric citrate were determined to enter via phagocytosis. By comparison, USPIO particles coated with the polymer carboxydextran enter cells by pinocytosis. For more information about these new labeling techniques, please see the references below, or contact our technical assistance department at techservice@markergene.com.

• Bloem JL, Wondergem J, (1989) "Gd-DTPA as a contrast agent in CT." Radiology 171(2):578-9.
• Pratten MK and Lloyd JB, (1986) "Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro." Biochim. Biophys. Acta 881: 307–313.
• Metz, S, Bonaterra G, Rudelius M, Settles M, Rummeny E, Daldrup-Link H, (2004) "Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro." Eur. Radiol. 14: 1851-1858.
• Zelivyanskaya ML, Nelson JA, Poluektova L, Uberti M, Melissa Mellon M, Gendelman HE, Boska MD, (2003) Tracking superparamagnetic iron oxide labeled monocytes in brain by high-field magnetic resonance imaging. J. Neurosci. Res. 73: 284-295.
• Shapiro EM, Skrtic S, Koretsky AP, (2005) Sizing it up: cellular MRI using micron-sized iron oxide particles. Magn. Reson. Med. 53: 329–338
• Wu YL Yi, Q, Foley, LM, Hitchens, K, Sato, K, Williams, JB, Ho C, (2006) " In situ labeling of immune cells with iron oxide particles: an approach to detect organ rejection by cellular MRI." Proc. Natl. Acad. Sci. USA 103: 1852–1857
• de Vries IJ, Lesterhuis WJ, Barentsz JO, Verdijk P, van Krieken JH, Boerman OC, Oyen WJG, Bonenkamp JJ, Boezeman JB, Adema GJ, Bulte JWM, Scheenen TWJ, Punt CJA, Heerschap A, Figdor CG, (2005) "Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy." Nat. Biotechnol. 23: 1407–1413.

Bronchoalveolar Eosinophil Staining with FITC.
White blood cells (WBCs) are a major component of the body's immune system. White blood cells are composed of granulocytes (neutrophils, eosinophils, and basophils) and non-granulocytes (lymphocytes and monocytes). Eosinophil granulocytes, commonly referred to as eosinophils (or less commonly as acidophils), are white blood cells of the immune system that are responsible for combating infection or parasites in vertebrates. They also control the immune mechanisms associated with allergy and asthma. These granulocytes develop in the bone marrow before migrating into the blood stream. An absolute eosinophil count is a blood test that measures the total number activated esoinophils in the white blood cell count in a blood sample. Eosinophils become active in certain allergic diseases, infections, and other medical conditions. The eosinophil count, therefore, can be an indication of disease or infection.

The eosinophil cells are normally transparent, but appear brick-red when stained with the dye eosin, using the Romanowsky method. The staining is concentrated in small granules within the cellular cytoplasm, which contain many chemical mediators, such as histamine and proteins such as eosinophil peroxidase, RNase, DNases, lipase and plasminogen. These mediators are released by a process called degranulation following activation of the eosinophil, and are toxic to both the parasite or infected host tissues. Eosinophils can also be easily detected by labeling them with FITC. They have over 100- fold higher affinity to bind FITC compared with other blood cells. Exposure of formaldehyde or ethanol- fixed cells to 2 – 500 nM of FITC in PBS labels essentially only eosinophils. Needless to say, FITC is several orders of magnitude less expensive than various antibodies used to identify these cells. Eosinophils can also be characterized by their blue autofluorescence upon UV excitation. The FITC dye can therefore be used  to discriminate eosinophils in a bronchoalveolar lavage sample by flow cytometry. For more information about these techniques for staining and analysis of eosinophils, please see the references below or visit our website.

• Bedner E, Halicka HD, Cheng W, Salomon T, Deptala A, Gorczyca W, Melamed MR. Darzynkiewicz, Z, (1999) "High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry. A potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry." Cytometry 36:77-82.
• Lendrum, AC, (2005) " The staining of eosinophil polymorphs and enterochromaffin cells in histological sections." J. Path. Bacteriol. 56(3): 441-441.
• Berretty, PJM, Cormane, RH, (1978) "The aniline blue fluorescence staining of eosinophilic granulocytes" Brit. J. Dermatology 99(4): 377-382.
• Horobin RW, Walter KJ, (1987) "Understanding Romanowsky staining." Histochem. Cell Biol. 86(3): 331-336.

a-Mannosidase Measurement in Mucolipidosis and Dystrophy.
Deficiency of lysosomal a-mannosidase is the primary defect in a-mannosidosis, a disease that leads to storage of various oligosaccharides that have an a-mannose residue at their non-reducing terminals. a-Mannosidase is elevated in plasma from patients with mucolipidosis type II and mucolipidosis type III, pseudo-Hurler polydystrophy and several other dystrophies, and measurement of the enzyme in amniotic fluid is useful for prenatal diagnosis of these disorders. These diseases are exemplified by progressive psychomotor retardation and deafness. Death usually occurs by 5 years of age. There are no known therapies to treat this disease. A bone marrow transplant in a patient with type 1 mannosidosis was not successful in restoring enzyme activity in brain tissue. Serum concentrations of a-mannosidase and beta-hexoaminidase have also been found to be significantly increased in patients with HIV CDC III and CDC IV without correlation to CD4+ count and secondary infection.

Several methods have been reported to measure enzyme activities in clinical and research samples. Hydrolysis of the synthetic substrate 4-methylumbelliferyl-a-D-mannopyranoside at acid pH (3-4) followed by measuring the fluorescence of the liberated 4-methylumbelliferone after stopping the reaction with an alkaline buffer system is the most common method used thusfar for analysis. A similar colorimetric assay has been developed using the substrate p-nitrophenyl a-D-mannopyranoside. But since only class II mannosidases appear to be active towards aryl mannosides, most serum mannosidases that have been described thusfar must be regarded as class II mannosidases. Class I α-mannosidases specifically cleave α-1,2-mannose linkages in a Ca2+-dependent manner, but are inactive towards artificial substrates like aryl mannosides. These enzymes are type II integral membrane proteins that are specifically retained within the endoplasmic reticulum and the Golgi complex. Class I α-mannosidases play an essential role in N-glycan processing during glycoprotein biosynthesis in mammalian cells, by removing up to four mannose residues from the high-mannose core structure Man9GlcNAc2 to form elaborated glycans. Marker Gene is currently working with researchers at several institutions to develop new a-mannosidase assay systems for both class I and class II enzymes. For more information on these methods, please see the references below, or visit our website.

• Lugering N, Stoll R, Kucharzik T, Busch H, Domschke W. (1994) " High levels of beta-hexoaminidase and a-mannosidase in sera of HIV+ individuals." Int Conf AIDS. 10: 157 (abstract no. PB0053).
• Ockerman PA (1967) A generalized storage disorder resembling Hurlers syndrome. Lancet II:239–241.
• Monus Z, Konyar E, Szabo L (1977) Histomorphologic and histochemical investigations in mannosidosis, a light and electron microscopic study. Virchows Arch B Cell Pathol 26:159–173.
• Beaudet L, Thomas GH (1989) Disorders of glycoprotein degradation. In: Scriver CR, Beaudet AL, Sly WS, Valle D
  (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, New York, pp 1603–1621.
• Cooper WA, Hatton C, Sardharwalla IB, Evans DI, Stevens RF (1987) Bone marrow transplantation in the treatment of alpha-mannosidosis. Arch Dis Child 62:1044–1049.
• Porwoll, S, Fuchs H, Tauber R, (1999) "Characterization of a soluble class I α-mannosidase in human serum " FEBS Lett. 449(2-3):175-178.

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays - High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Marker Gene Accepts Major Credit Cards.
Place your orders now, using Master Card or Visa and save time and money! Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only). We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960. International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering. For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com. We will be happy to assist you.