Is ataxic gait the predominant presenting manifestation of Creutzfeldt-Jakob disease? Experience of 14 Chinese cases from Taiwan

The Cre recombinase of bacteriophage P1 catalyses site-specific recombination between lox-recombination target sites both in prokaryotic and eukaryotic cells and has thus become a popular tool in genetic research. Stable, Cre-mediated integration of DNA sequences at pre-existing lox sites in the eukaryotic genome is facilitated when a Cre recombinase protein rather than a cre-expression plasmid is used to direct site-specific recombination (Baubonis and Sauer (1993) Nucleic Acids Res., 21, 2025-2029). We bacterially produced a Cre recombinase containing a nuclear localisation signal as a fusion protein with the E. coli maltose binding protein (MBP) and purified the protein by one step affinity chromatography. Subsequent cleavage with the protease factor Xa releases the Cre recombinase including the nuclear localisation signal from the maltose binding protein. Surprisingly, we found that the recombination activity of the uncleaved MBP-Cre fusion protein is virtually identical to that of the native Cre recombinase. This suggests that the MBP portion of the fusion protein behaves as a separate protein domain which does not interfere with Cre activity and can thus be used as an independent molecular tag. Additionally, the fusion protein is very resistant to proteolytic degradation and active over a wide range of temperatures. It efficiently catalyses excision and integration reactions in vitro and in eukaryotic cells. Finally, we could show that, by using MBP-Cre, it is possible to concomitantly excise a lox-flanked DNA sequence from a plasmid and integrate it into a pre-existing lox site in the genome in one transfection experiment. Vector backbone sequences which might have undesirable effects can thereby be excluded. The MBP-Cre fusion protein described here will be a useful tool not only for the catalysis of Cre-mediated recombination reactions in vitro and in vivo but also for the analysis of the mechanism of site-specific recombination.     

Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion

Vitronectin (VN), a major cell adhesion protein, is found in plasma and in the extracellular matrix. At least three distinct cell surface receptors for vitronectin belonging to the integrin superfamily have been identified in normal and neoplastic cells. Many cell adhesion ligands, including vitronectin, contain an Arg-Gly-Asp (RGD) sequence mediating, in part, the ligand-receptor interaction. These ligands bind different integrins with varying specificity and affinity. The mechanism of receptor specificity remains controversial. To determine the role of the RGD sequence in receptor specificity, we amplified the cDNA for human vitronectin from a liver cDNA library and generated two separate mutants by utilizing site-directed mutagenesis resulting in aspartic acid (Asp47) to glutamic acid (Glu47) substitution and glycine (Gly46) to alanine (Ala46) substitution. The mammalian expression vector, pZEM229R, was used to transfect baby hamster kidney cells which secreted recombinant proteins into the supernatant. All recombinant proteins were isolated by heparin-agarose chromatography and tested for interaction with three known vitronectin receptors, namely, alpha IIIb beta 3 on thrombin-activated platelets, alpha v beta 3 on human umbilical vein endothelial cells and alpha v beta 5 on Panc-1 cells. Recombinant wild-type vitronectin behaved in a fashion similar to plasma-derived vitronectin. Both the RGE-VN and RAD-VN recombinant mutant proteins showed complete loss of cell adhesion activity, regardless of the receptor. These results confirm the essential and central role of the RGD sequence in vitronectin for cell adhesion. This expression system allows further structure/function analysis of vitronectin.     

Early postischemic dantrolene-induced amelioration of poly(ADP-ribose) polymerase-related bioenergetic failure in neonatal rat brain slices

In the infant brain, ischemia-induced ionic and enzyme mechanisms may independently lead to cell death by energy depletion: resequestration of calcium mobilized from intracellular stores consumes ATP, and activated poly(ADP-ribose) polymerase (PARP) uses oxidized nicotinamide adenine dinucleotide to form polyADP-ribosyl nuclear proteins associated with DNA damage. Using 31P nuclear magnetic resonance spectroscopy, we have monitored intracellular pH and cellular energy metabolites in ex vivo neonatal rat cerebral cortex before, during, and after substrate and oxygen deprivation. In an insult that exhibited secondary energy failure and apoptosis we identified a relative 25% augmentation of high-energy phosphates at the end of recovery when the ryanodine-receptor antagonist, dantrolene, was introduced in the early (0- to 40-minute) but not late (40- to 120-minute) stage of recovery (P < 0.05). In contrast to the absence of a late dantrolene-sensitive effect, inhibition of PARP with 3-methoxybenzamide was as effective (P < 0.05) as early dantrolene, even when introduced after a 40-minute delay. The dantrolene and 3-methoxybenzamide effects on high-energy phosphates were not additive, rather the early dantrolene-sensitive effect nullified the potential 3-methoxybenzamide effect. Therefore, in this vascular-independent neonatal preparation, postischemic mobilization of calcium from intracellular stores is associated with PARP-related energy depletion. Inhibition of either of these processes confers improved postischemic bioenergetic recovery in the developing brain.     

Quality assessment of intraoperative blood salvage and autotransfusion

Intraoperative blood salvage and autotransfusion are commonly used to minimize exposure to banked blood. Although this technique has been used widely for years, data vary regarding the quality of autotransfused blood. Salvaged blood may contain plasma, residual heparin, and free hemoglobin released from damaged cells. All of these factors may contribute to the adverse sequelae sometimes seen with autotransfusion. For these reasons, we have monitored autotransfused blood to assess its quality. Intraoperative blood salvage was used during most cardiac procedures and at the discretion of the surgeon in other specialties. Blood was collected through a double lumen catheter that was anticoagulated with heparin, filtered, centrifuged, and washed with saline. A sample of the blood was removed for analysis, which included hematocrit, heparin assay, fibrinogen, and free hemoglobin levels. Over a 6-year period, 1593 patients had intraoperative blood salvage with quality assessment. The majority of patients underwent cardiac operations (941 patients, 59%), whereas 243 had orthopedic (15%) and 208 had vascular (13%) procedures. Additionally, there were 127 pediatric patients (8%) and 74 miscellaneous procedures (5%). The highest average yield of salvaged blood was during vascular procedures (1073 +/- 76 mL), whereas orthopedic cases had the lowest yield (378 +/- 19 mL) and hematocrit (39%). There was minimal residual heparin activity, even in patients requiring systemic anticoagulation (0.3 to 0.5 units/mL). Patients undergoing pediatric procedures had the lowest concentration of free hemoglobin (476 mg/L), whereas all adult patients had higher free hemoglobin levels, especially vascular operations (990 mg/L). Intraoperative salvaged blood has minimal heparin activity, even in procedures requiring systemic anticoagulation. Fibrinogen, a marker of residual plasma, was undetectable in the majority of cases. These data indicate that intraoperative blood salvage generally results in a high-quality product (good hematocrit, low heparin, minimal plasma), although there are significant differences in free hemoglobin levels depending on the operative procedure.     

The IPCS Collaborative Study on Neurobehavioral Screening Methods: V. Results of chemical testing. Steering Group

The goal of the International Programme on Chemical Safety (IPCS) Collaborative Study on Neurobehavioral Screening Methods was to determine the intra- and inter-laboratory reliability of a functional observational battery (FOB) and an automated assessment of motor activity in eight laboratories worldwide. The control data were crucial to the outcome of the studies in terms of sensitivity and reliability of the test measures, which in turn impact on the between-laboratory comparisons of chemical effects. In addition, analyses of control data can aid in determining endpoints that may require modification to improve their sensitivity and reliability. The control data from the eight laboratories were examined in terms of the following parameters: 1) control variability within studies for each laboratory; 2) within-laboratory replicability of control values across studies; 3) within-laboratory stability of control values over the course of testing for a given study; and 4) between-laboratory comparisons of parameters (1), (2), and (3). The analyses indicated considerable differences across endpoints, wherein some measures showed high variability and little replicability, while others were extremely reproducible. Generally, there were similar ranges of variability and replicability of control data across laboratories, although in some cases one or two laboratories were markedly different from the others. The physiological (weight, body temperature) and neuromuscular (grip strength, landing foot splay) endpoints exhibited the least variability, whereas the subjective assessments of reactivity varied the most. These data indicate a reasonable degree of comparability in the data generated in the participating laboratories.     

Susceptibilities of Legionella spp. to newer antimicrobials in vitro

The in vitro activities of 13 antimicrobial agents against 30 strains of Legionella spp. were determined. Rifapentine, rifampin, and clarithromycin were the most potent agents (MICs at which 90% of isolates are inhibited [MIC90s], < or = 0.008 microgram/ml). The ketolide HMR 3647 and the fluoroquinolones levofloxacin and BAY 12-8039 (MIC90s, 0.03 to 0.06 microgram/ml) were more active than erythromycin A or roxithromycin. The MIC90s of dalfopristin-quinupristin and linezolid were 0.5 and 8 micrograms/ml, respectively. Based on class characteristics and in vitro activities, several of these agents may have potential roles in the treatment of Legionella infections.     

Gastric graft-versus-host disease: a blinded histologic study

Acute graft-versus-host disease (GvHD) of the upper gastrointestinal (GI) tract is common after allogeneic bone marrow transplantation (BMT). However, diagnosis cannot be made on clinical presentation and endoscopic findings alone, because these are nonspecific, and histologic confirmation is often desirable. The diagnosis of gastric GvHD is often based on subtle findings with considerable potential for variability in interpretation. Evaluation of the reproducibility of diagnosis and recognition of histologic features of gastric GvHD was based on blinded review of 56 gastric biopsies (24 from patients with allogeneic BMT or unrelated umbilical cord blood transplantation and 32 control biopsies from patients who did not undergo BMT, of whom eight had active GI cytomegalovirus [CMV] infection). Histologic criteria for GvHD were apoptosis and gland destruction, sparse inflammatory infiltrate, and granular eosinophilic debris in dilated glands. Seventeen patients (22 biopsies) were judged to have clinical GvHD on the basis of skin or liver involvement and GI symptoms without other known cause. Eighteen of these 22 gastric biopsies were classified as GvHD by at least two of the three pathologists on initial review. Blinded histologic diagnosis of GvHD had a positive predictive value of 69%, a sensitivity of 82%, and specificity of 76%. False-positive results occurred in CMV gastritis, human immunodeficiency virus (HIV) infection, primary immunodeficiency, and after renal transplantation. Of individual features, granular debris in glands was a specific (94% specificity), but insensitive (41% sensitivity) marker for GvHD. Distinction between GvHD and CMV infection can be difficult, and GvHD can be confused with changes seen in HIV infection and other immunodeficiency states.     

Cerebellar nitric oxide synthase is expressed within granule cell patches innervated by specific mossy fiber terminals: a developmental profile

The nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining technique was utilized as a marker of nitric oxide synthase (NOS) to map NOS expression in developing and adult rat cerebellum. NADPH-d-positive cells were first visualized in the cerebellar cortex at postnatal day 5 (PND5) which increased to peak levels by PND30 when they began to exhibit a patch-like organization. In order to determine the relationship of the NADPH-d staining pattern with mossy fiber innervation, mossy fiber projections were traced using cholera toxin B subunit or biocytin injected into the lateral reticular nuclei (LRtN) or pontine nuclei (PtN), respectively. Double staining revealed that the clustered mossy fiber terminals projecting from the ventrorostral LRtN and caudal PtN were well matched with NADPH-d-stained patches. This patch-like localization of NOS matched with specific mossy fiber terminals in adult cerebellum implicates these NOS patches as defining distinct anatomical zones.     

Measuring intraocular pressure in keratoconus. Effect of the changed biomechanics]

Forty-two team orthopedists representing all 28 major league baseball teams were surveyed to ascertain their definitive treatment for a hypothetical starting rotation pitcher who had sustained a grade III acromioclavicular (AC) separation to his throwing arm 1 week before the season. Twenty-nine (69%) of the physicians would treat the injury nonoperatively, while 13 (31%) would operate immediately. Twenty-five (60%) of the orthopedists had actually treated a pitcher or position baseball player with a grade III AC separation in the throwing arm, the 25 treating a total of 32 patients. Twenty (63%) of these injuries were treated nonoperatively, and 12 (37%) were treated operatively. The physicians reported that 16 (80%) of the patients treated nonoperatively regained normal function and achieved complete relief of pain, while 18 (90%) had normal range of motion after treatment; of those treated operatively, 11 (92%) regained normal function, achieved complete relief of pain, and had normal range of motion after surgery.