Amplification of Ehrlichial DNA from Dogs 34 Months after Infection with Ehrlichia canis

In order to determine whether dogs in the subclinical phase of canine monocytic ehrlichiosis (CME) are carriers of Ehrlichia canis and to determine the significance of persistent indirect immunofluorescent anti-E. canis antibody titers during this phase, PCR was performed with blood, bone marrow, and splenic aspirates collected 34 months postinoculation from six clinically healthy beagle dogs experimentally infected with E. canis. At least one of the three samples (spleen, bone marrow, and blood) from four of the six dogs was PCR positive. The spleens of all four of these dogs were PCR positive, and the bone marrow and blood of two of the four dogs were PCR positive. Indirect immunofluorescent-antibody titers increased progressively during the first 5 months postinfection, remained high for an additional period of more than 11 months, and declined thereafter, suggesting that the dogs were recovering from the disease. Five of the dogs remained seropositive 34 months postinfection. The data obtained in this study demonstrate for the first time that clinically healthy dogs in the subclinical phase of CME are carriers of the rickettsia. It was shown that dogs can harbor E. canis for years without developing the chronic clinical disease and that dogs can eliminate the parasite and recover from CME without medical treatment. Our findings suggest that the spleen is the organ most likely to harbor E. canis parasites during the subclinical phase and the last organ to accommodate the parasite before elimination. It was concluded that PCR of DNA extracted from splenic aspirates is a reliable method for determining the carrier state of CME.     

Molecular mechanism of kNBC1–carbonic anhydrase II interaction in proximal tubule cells

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L⁹⁵⁸DDV and D⁹⁸⁶NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.     

Preparation, characterization, and performance of magnetic iron-carbon composite microparticles for chemotherapy

Magnetic microcarrier particles useful for delivering chemotherapeutic drug molecules are described. The particles are formed by joint deformation of iron and carbon in a ball mill. Physical, chemical, and functional characterization has been carried out on the particles. Physical characteristics include microscopy, particle size analysis (0.5-5 microm), surface area (250 m2/g), water vapor adsorption isotherm (hydrophobic surface), and analysis of the iron-carbon interface by M?ssbauer spectroscopy, X-ray diffraction, and differential thermal analysis. Chemical analysis was used to identify elements in the particles other than carbon and iron. Functional characteristics measured included the particles' ability to adsorb and desorb doxorubicin, cytotoxicity, and their magnetic susceptibility.     

Transplantation of hematopoietic stem cells for induction of unresponsiveness to organ allografts

Although it has been recognized since the early days of Owen and Medawar that engraftment of donor stem cells, induced in utero spontaneously or intentionally neonatally, results in life-long unresponsiveness to donor alloantigens. However, successful induction of transplantation tolerance in adult life still represents an unsolved problem. Engraftment of donor stem cells using conventional modalities involves intensive myeloablative or lymphoablative immunosuppression, which is associated with toxicity and mortality and such methods are not suitable for organ allograft recipients. In this chapter, we present an innovative approach for induction of donor-specific unresponsiveness to bone marrow and organ allografts without myeloablative conditioning. Our methods is based on cyclophosphamide-induced, alloantigen-primed lymphocyte depletion. Cyclophosphamide is administered 1 day following infusion of donor hematopoietic cells, thus eliminating predominantly host T lymphocytes reacting against donor cell challenge, and resulting in relative unresponsiveness to donor alloantigens. Subsequently, life-long tolerance to fully mismatched donor skin allografts can be accomplished by a second infusion of stem cells from the same donor, with donor T cells displacing residual alloreactive host cells that may have escaped deletion. Taken together, we believe that induction of true permanent and specific tolerance to organ allografts using donor hematopoietic cells could become a clinical reality in the foreseeable future.     

Ca2+ clearance in visual motion-sensitive neurons of the fly studied in vivo by sensory stimulation and UV photolysis of caged Ca2+

In motion-sensitive visual neurons of the fly, excitatory visual stimulation elicits Ca(2+) accumulation in dendrites and presynaptic arborizations. Following the cessation of motion stimuli, decay time courses of the cytosolic Ca(2+) concentration signals measured with fluorescent dyes were faster in fine arborizations compared with the main branches. When indicators with low Ca(2+) affinity were used, the decay of the Ca(2+) signals appeared slightly faster than with high affinity dyes, but the dependence of decay kinetics on branch size was preserved. The most parsimonious explanation for faster Ca(2+) concentration decline in thin branches compared with thick ones is that the velocity of Ca(2+) clearance is limited by transport mechanisms located in the outer membrane and is thus dependent on the neurite's surface-to-volume ratio. This interpretation was corroborated by UV flash photolysis of caged Ca(2+) to systematically elicit spatially homogeneous step-like Ca(2+) concentration increases of varying amplitude. Clearance of Ca(2+) liberated by this method depended on branch size in the same way as Ca(2+) accumulated during visual stimulation. Furthermore, the decay time courses of Ca(2+) signals were only little affected by the amount of Ca(2+) released by photolysis. Thus Ca(2+) efflux via the outer membrane is likely to be the main reason for the spatial differences in Ca(2+) clearance in visual motion-sensitive neurons of the fly.     

Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.     

Comparative Carcinogenicity of 5,5-Diphenylhydantoin with or without Perinatal Exposure in Rats and Mice

Volume 21, Number 2 (1993), in the article "Comparative Carcinogenicityof 5,5-Diphenyihydantoin with or without Perinatal Exposurein Rats and Mice," by R. S. Chhabra, J. R. Bucher, J. K. Haseman,M. R. Elwell, P. J. Kurtz, and B. D. Canton, pages 174–186:Since the publication of the above article, the authors havefound that the feed and estimated DPH consumption data reportedin Tables 2 and 6 were incorrect. The following Tables 2 and6 contain revised feed and DPH consumption data:     

Permanent Cardiac Pacing in Octogenarians

A significant percentage of patients in need of a permanent pacemaker are older than 80 years. The implantation policy may be determined either by the patient's physical activity or by chronologic age. The trend in pacemaker implantation in patients over 80 during the last 10 years in our institution was evaluated and compared with the trend in the patients younger than 80 at the time of implantation. Of 519 patients who had primary pacemaker implantation, 152 (29%) were older than 80 at the time of the procedure. Another 189 patients had second implantation procedures, and 80% of them were older than 80 years. Complete atrioventricular block was the indication for pacing in 44 ?+/- 11% and sick sinus syndrome in 25 ?+/- 7%. The tendency to implant dual-chamber pacemakers increased from 0% during 1985 to 76% in 1994, including 69% DDD and 31% DDDR, but the transition was faster in the younger group. By 1994, there was no difference in the incidence of advanced pacing systems in the 2 age groups. During 1985, only VVI pacemakers were replaced, and during 1994, less than 10% were replaced with simple ventricular pacing units. Pacing system upgrading was frequent during the second half of the decade. The success and complication rate of implantation did not differ in the 2 groups.