Testing for Mycobacterium avium subsp. paratuberculosis infection in asymptomatic free-ranging tule elk from an infected herd

Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp. paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three Johne's disease assays: enzyme linked immunosorbent assay (ELISA); agar gel immunodiffusion assay (AGID), and fecal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of confirmed infection, eight had elevated ELISA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mptb infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals.     

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.     

Impaired stimulation of intestinal glucose absorption by portal insulin via hepatoenteral nerves in chronically ethanol-intoxicated rats

In the isolated, jointly perfused small intestine and liver of rats insulin, infused into the portal vein, induced an increase in intestinal glucose absorption via hepatoenteral cholinergic nerves. The possible loss of function of these nerves due to ethanol-induced neuropathy was investigated with 6 weeks ethanol-fed rats. Portal insulin or arterial carbachol failed to increase intestinal glucose absorption but cAMP still did so. The intact stimulatory effect of cAMP indicated an undisturbed capacity of the enterocytes. The loss of action of portal insulin and of arterial carbachol can be explained by the impairment of the hepatoenteral nerves in line with an ethanol-induced neuropathy.     

Optical recording system based on a fiber optic image conduit: assessment of microscopic activation patterns in cardiac tissue.

Optical recording of transmembrane voltage changes with the use of potentiometric dyes has opened the possibility of determining spatial patterns of electrical activity in excitable tissues. To follow such activation patterns on the cellular/subcellular level in heart cell cultures, a recording system was developed that features both high spatial resolution (4-200 microm) and high temporal resolution (uncertainty in the determination of delays between fast rising signals of +/-1 micros). Central to the system is a fiber optic image conduit consisting of 379 individual optical fibers. At one end the fibers are fused to form an input window that matches the size of the field of view of the microscope. At the other end, the fibers are loose, permitting a selectable subset to be connected to 80 discrete photodetectors. This design allows the sensitive area of the imager to be adapted to regions of interest in a given preparation, thus making optimal use of the limited number of detectors. Furthermore, by using a second fiber optic imager, individual photodetectors can be assigned to different optical ports, thus providing the means for fast and simultaneous dual-emission wavelength measurements. This feature permitted the elimination of motion artifacts arising from the myocytes without the use of contraction-suppressing drugs.     

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2,2-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 M gemcitabine for 8 h, or staurosporine (1.0 M) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 M) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 M gemcitabine increased phosphatidylserine expression in a small fraction of cells (5–10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 M staurosporine or 10 M gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.     

Synthesis and antimycobacterial properties of N-substituted 6-amino-5-cyanopyrazine-2-carboxamides

A series of fifteen new compounds related to pyrazinamide (PZA) were synthesized, characterized with analytical data and screened for antimycobacterial, antifungal and antibacterial activity. The series consists of 6-chloro-5-cyanopyrazine-2-carboxamide and N-substituted 6-amino-5-cyanopyrazine-2-carboxamides, derived from the previous by nucleophilic substitution with various non-aromatic amines (alkylamines, cycloalkylamines, heterocyclic amines). Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis equal to pyrazinamide (12.5-25 μg/mL). More importantly, 6-chloro-5-cyanopyrazine-2-carboxamide and 5-cyano-6-(heptylamino)pyrazine-2-carboxamide were active against Mycobacterium kansasii and Mycobacterium avium, which are unsusceptible to PZA. Basic structure-activity relationships are presented. Only weak antifungal and no antibacterial activity was detected.