Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice.

Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinson's disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.

DNAJC13 p.Asn855Ser mutation screening in Parkinson's disease and pathologically confirmed Lewy body disease patients.

BACKGROUND: Recently, a novel mutation in exon 24 of DNAJC13 gene (p.Asn855Ser, rs387907571) has been reported to cause autosomal dominant Parkinson's disease (PD) in a multi-incident Mennonite family. METHODS: In the present study the mutation containing exon of the DNAJC13 gene has been sequenced in a Caucasian series consisting of 1938 patients with clinical PD and 838 with pathologically diagnosed Lewy body disease (LBD). RESULTS: Our sequence analysis did not identify any coding variants in exon 24 of DNAJC13. Two previously described variants in intron 23 (rs200204728 and rs2369796) were observed. CONCLUSION: Our results indicate that the region surrounding the DNAJC13 p.Asn855Ser substitution is highly conserved and mutations in this exon are not a common cause of PD or LBD among Caucasian populations.

Direct measurement of species-specific cohesion in cellular slime molds.

Partially differentiated cells of two species of cellular slime molds, Dictyostelium discoideum and Dictyostelium purpureum, were labeled with isothiocyanate derivatives of fluorescent dyes. The labeled cells of each species segregated promptly when mixed and placed on moist filters. We determined whether cells studied at a time when they demonstrated this capacity to segregate showed a preferential adherence to cells of the same species. When labeled dissociated cells of each species were interacted with an unlabeled immobilized layer of cells of each species under appropriate conditions, binding was, in part, species-specific.

Cell adhesion molecules: detection with univalent second antibody.

Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against certain cell surface molecules of Dictyostelium discoideum blocks cell-cell adhesion when the in vitro assay is performed in the presence of univalent goat anti-rabbit antibody. Under appropriate experimental conditions, the univalent second antibody blocks agglutination induced by the rabbit antibody without significantly interfering with its effect on cell-cell adhesion. This method promises to be useful for screening monoclonal antibodies raised against potential cell adhesion molecules because: (a) it allows for the screening of large numbers of antibody samples without preparation of univalent fragments; and (b) it requires much less antibody because of the greater affinity of divalent antibodies for antigens.

Endogenous cell surface lectin in Dictyostelium: quantitation, elution by sugar, and elicitation by divalent immunoglobulin.

The amount of total endogenous cellular and cell surface lectin in aggregating Dictyostelium purpureum was determined by a number of immunochemical techniques. The results show that of the 5 x 10(6) molecules of the lectin (called purpurin) per aggregating cell only about 2% (1 x 10(5) molecules) is present on the cell surface. Cell surface purpurin can be specially eluted by lactose, which indicates that it is held to the surface by its carbohydrate-binding site. The eluted purpurin is replaced on the cell surface within 45 min. Estimates of cell surface purpurin made by binding of specific immunoglobulin to the cells at 4 degrees C indicate that a much larger amount, about 1 x 10(6) molecules, becomes associated with the cell surface in the presence of this divalent ligand. In contrast, univalent antibody fragments do not have this effect.

Waldenstrom macroglobulinemia cells devoid of BTKC481S or CXCR4WHIM-like mutations acquire resistance to ibrutinib through upregulation of Bcl-2 and AKT resulting in vulnerability towards venetoclax or MK2206 treatment.

Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9-3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTKC481S and CXCR4WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTKC481S and CXCR4WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.

NBR1 is dispensable for PARK2-mediated mitophagy regardless of the presence or absence of SQSTM1.

Degradation of malfunctional mitochondria by mitophagy is a pivotal component of mitochondrial quality control to maintain cellular homeostasis. Mitochondrial clearance through the PINK1/PARK2 pathway is mediated by autophagic adaptor proteins. Previous studies revealed a significant involvement, but not an absolute requirement for SQSTM1 in PARK2-dependent mitophagy, suggesting that the existence of redundant adaptor proteins may compensate for the loss of SQSTM1. Here we investigated whether NBR1, a functional homolog of SQSTM1, has a role in PARK2-mediated mitophagy, either alone or as a compensatory mechanism. We showed that NBR1 does not appear to be required for mitochondrial clustering following mitochondrial depolarization. Moreover, we demonstrated that deletion of NBR1 alone or in combination with SQSTM1 does not prevent the degradation of damaged mitochondria. Our data suggest that NBR1 is dispensable for PARK2-dependent mitophagy and additional autophagic adaptor proteins, other than NBR1, are responsible for mitochondrial degradation in cells depleted of SQSTM1.

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli.

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.

Dispensable sequences and packaging constraints of DNA from the Streptomyces temperate phage phi C31.

Deletion mutants of the temperate Streptomyces phage phi C31 were selected by two methods: resistance to the chelating agent sodium pyrophosphate, and plating of a phi C31::pBR322 hybrid phage on Streptomyces albus G to obtain large plaque mutants. The deletions defined a 7.7 kilobase (kb) segment of the phi C31 genome which is inessential for plaque formation, in addition to a shorter segment including the repressor gene. Analysis of deletions and insertions suggested that the minimum size of the phi C31 genome allowing plaque formation is 37.5 kb (91% of the wild-type length of 41.2 kb), and the maximum is at least 42.4 kb (103%). These results indicate that it should be possible to introduce up to 10 kb of foreign DNA into a suitably developed phi C31 vector.

Evaluation of a new reagent for preserving fresh blood samples and its potential usefulness for internal quality controls of multichannel hematology analyzers.

We describe a new, easy-to-use reagent, Cyto-Chex (Streck Laboratories, Omaha, Neb), that preserves fresh whole blood in a non-cross-linking, nonformalin manner. Target values assigned to fresh blood were essentially met after preservation and storage of up to 31 days. Respective mean analytic inaccuracies and short-term intra-assay coefficients of variation (n = 30) were as follows: WBCs, 6.7% and 1.99%; RBCs, 0.7% and 0.76%; hemoglobin, -1.8% and 0.79%; hematocrit, -0.3% and 0.75%; mean corpuscular volume, -1.0% and 0.78%; and platelets, 6.9% and 3.12%. Linearity of dilution-sensitive analytes was satisfactory over a wide range of dilutions after preservation of blood samples. Ten independent laboratories using 10 different instruments determined day-to-day interassay imprecision during four 7-day periods after blood preservation. Mean interassay coefficients of variation for participating laboratories were WBC, 1.92%; RBC, 1.00%; hemoglobin, 1.29%; hematocrit, 2.00%; and platelets 3.29%. Cyto-Chex enables long-term monitoring of instrument accuracy and precision with retained blood specimens of healthy persons. Blood from patient cohorts with various hematologic disorders and with a wide range of numeric abnormalities and/or parameter aberrations can be preserved satisfactorily with this reagent. The reanalysis of preserved patient blood samples is an important adjunct to the use of commercial control material in quality control programs of multichannel hematology analyzers.

Improved left ventricular function after chronic left ventricular unloading.

BACKGROUND: This study assessed the effect of prolonged left ventricular unloading on native ventricular function. METHODS: We reviewed data from 31 patients (30 men, 1 woman) supported more than 30 days (mean, 137 days; range, 31 to 505 days) with the HeartMate left ventricular assist system. The patients' mean age was 46 years (range, 22 to 64 years); 17 had idiopathic and 14 had ischemic cardiomyopathy. Data (anatomic, physiologic, hemodynamic, histologic, and biochemical) were collected at the time of HeartMate implantation, during support with the device temporarily off, and at the time of device explantation. RESULTS: Routine chest roentgenogram showed improvement in cardiothoracic ratio (0.62 +/- 0.04 to 0.55 +/- 0.03; p < 0.0001). Echocardiography performed with the pump off showed a significant decrease in left ventricular end-diastolic dimension (6.81 +/- 0.87 cm to 5.39 +/- 1.08 cm; p < 0.0005) and a significant improvement in ejection fraction (0.11 +/- 0.05 to 0.22 +/- 0.17; p < 0.02). Cardiac index increased (1.96 +/- 0.52 L.min-1.m-2 to 2.93 +/- 0.73 L.min-1.m-2; p < 0.0001), mean aortic pressure increased (71.40 +/- 10.63 mm Hg to 76.33 +/- 16.84 mm Hg; p = 0.48), pulmonary capillary wedge pressure decreased (24.18 +/- 6.27 mm Hg to 14.48 +/- 3.01 mm Hg; p < 0.0001), and pulmonary vascular resistance decreased (3.34 +/- 2.00 Wood units to 2.51 +/- 0.88 Wood units; p < 0.05). Comparisons of tissue samples taken at the time of implantation and at the time of transplantation showed a marked reduction in myocytolysis. Calcium uptake, calcium-binding rates, and lipid levels normalized in patients studied. Plasma norepinephrine levels decreased to near normal levels. CONCLUSION: Prospective studies are now indicated to determine whether device removal without transplantation may be beneficial in selected patients.

Prevalence of Mycoplasma and Chlamydia in the nasal flora of dairy calves.

Nasal swabs from dairy calves in 20 selected herds were collected once in each of three sampling periods in one year and cultured for mycoplasmas and chlamydiae. Seventy-seven of 411 calves (19%) and 16 of 20 herds (80%) were positive for mycoplasmas at least once throughout the year, and all of 378 calves sampled from the 20 herds were negative for chlamydiae. Thirty-one percent of the isolates were Mycoplasma bovirhinis; other species isolated less frequently were Mycoplasma bovis, Mycoplasma arginini, and Acholeplasma laidlawaii. There was no evidence of respiratory tract disease at the time of sampling in calves studied.

Revascularization procedures in patients with transplant coronary artery disease.

OBJECTIVE: To assess the efficacy of revascularization in cardiac transplant patients who developed de novo coronary artery disease. METHODS: Eighteen patients underwent one or more of four methods of revascularization: percutaneous transluminal coronary angioplasty (PTCA), percutaneous transluminal coronary rotational atherectomy (PTCRA), coronary artery bypass grafting (CABG), and transmyocardial laser revacularization (TMLR). Eleven PTCA procedures were performed in 10 patients 55.3 +/- 6.6 months after transplantation. Six patients underwent PTCRA 83.3 +/- 11.2 months after transplantation. Five patients underwent CABG 54.0 +/- 12.6 months after transplantation; the mean left ventricular ejection fraction was 49.6 +/- 16.9 (20-65%); hypertrophy was present in two of these patients. One patient with distal coronary artery disease and New York Heart Association class IV symptoms underwent TMLR only. One patient underwent both CABG and TMLR because of triple vessel proximal disease, diffuse distal disease, and New York Heart Association class IV symptoms. RESULTS: PTCA was successful in 10 procedures with decrease in mean stenosis from 87.7 +/- 2.7 to 24.3 +/- 6.0%. Follow-up, at 16.9 +/- 4.0 months, showed restenosis in two patients. PTCRA was successful in all patients with a decrease in mean stenosis from 83.4 +/- 4.4 to 11.7 +/- 1.9%. Short-term follow-up did not reveal reocclusion. Two CABG patients who had hypertrophy died of heart failure 2 and 9 days after their operations. One CABG patient with excellent cardiac function died after 15 days because of pulmonary failure. In one patient, left ventricular ejection fraction improved from 35 to 50%, and he is alive 64 months later. Six months after TMLR, the New York Heart Association class in one patient improved from IV to II, and his left ventricular ejection fraction improved from 29 to 42%. The ejection fraction in the patient who underwent both CABG and TMLR improved from 20 to 56% but the patient expired 7 weeks later. CONCLUSIONS: It appears that revascularization procedures can be effective in patients with coronary artery disease after cardiac transplantation and that coronary angioplasty or atherectomy would be a therapy of choice for single proximal lesions. CABG should be used cautiously and only reserved for patients with multi-vessel disease without hypertrophy. Laser revascularization with or without bypass grafting has potential to become the therapy of choice for transplant coronary artery disease.

Benign and malignant cells in effusions: diagnostic value of image DNA cytometry in comparison to cytological analysis.

Identifying tumor cells in body cavity fluids reliably is a well-known diagnostic problem. Since cytometric quantitation of nuclear DNA content appears to be a promising new tool in the diagnosis and prognostic evaluation of many solid human tumors, we examined its validity in detecting malignant cells in cytologically positive effusions. For this purpose, image DNA cytometric measurements, including the evaluation of DNA-ploidy and the calculation of the DNA index (DI), were performed in 80 body cavity fluids. The results were correlated with cytology, clinical course and final histological diagnoses. We used aneuploidy, as shown by interactive image DNA cytometry, as a marker for the malignancy of cells that occur in body cavity fluids with a 100% specificity and 94.8% sensitivity. Cytological investigation showed a 92.3% specificity and 95.4% sensitivity. Combining both methods raised the specificity to 100% and the sensitivity to 98.5% and had a positive predictive value of 100% and a negative predictive value of 93.8%. The DNA-index (DI) was significantly higher in malignant effusions than in benign effusions: 1.5 +/- 0.74 (mean +/- SD) versus 1.11 +/- 0.26 (p < 0.05). Along with the difficult cytological evaluation of malignant cells in body cavity fluids, image DNA cytometry can be a helpful additional method for evaluating these cells. Combining the two techniques results in a highly specific and sensitive prediction of malignant cells. We, therefore, suggest using these methods for the reliable identification of tumor cells in effusions.

Effect of reovirus infection and dietary levels of selected vitamins on immunocompetence of chickens.

The immunocompetence of 8-week-old reovirus WVU 2937-infected and uninfected chickens fed various dietary levels of biotin, niacin, choline, or folic acid was evaluated in four experiments. Antibody production to sheep red blood cells (SRBC), wattle responses to phytohemagglutinin-P (PHA-P), and wattle responses of tuberculin-sensitized chickens to bovine-purified protein derivative (PPD) were examined in five chickens per treatment. Dietary deficiencies had no effect on the in vivo T-cell immune responses to PPD, PHA-P, or humoral hemagglutinating-antibody production to SRBC. Reovirus-infected and uninfected chickens fed biotin at double the National Research Council (1977) requirement demonstrated decreased PPD responses. Lower hemagglutinating-antibody titers in the uninfected chickens and increased titers in the infected chickens were observed in those fed 200% of the requirement of biotin when compared with the chickens fed 20% and 100% biotin diets. Reovirus infection had no effect on PPD and PHA-P responses. However, in the choline experiment, reovirus-infected chickens had elevated titers. These data indicate that reovirus infection had no adverse effect on the immunocompetence of chickens 8 weeks postinfection. Dietary deficiencies also had no adverse effect on the immunocompetence of 8-week-old reovirus-infected and uninfected chickens.

A modified technique for assaying avian immunoglobulin-M-secreting cells.

A localized complement-dependent hemolytic assay in modified Cunningham chambers for enumerating avian immunoglobulin-M (IgM)-secreting spleen cells is defined. The maximum number of IgM-secreting spleen cells was present six days after sheep red blood cells (SRBCs) were injected. Intra-abdominal injection of SRBCs resulted in greater but more variable numbers of IgM-secreting cells at the time of maximum response than did either intramuscular or intravenous injection. Fresh chicken serum (FCS) with 10% guinea pig serum was an equally or more effective source of complement for the assay than was FCS alone.

Evaluation of secondary enrichment for detecting Salmonellae in bobwhite quail.

Secondary enrichment of cultures in tetrathionate-brilliant-green broth substantially increased Salmonella recovery over that achieved with primary tetrathionate-brilliant-green broth or primary selenite-cystine broth.

A comparison of avian and mammalian cell cultures for the propagation of avian reovirus WVU 2937.

Two avian and seven mammalian cell lines were evaluated for their application in propagating avian reovirus WVU 2937. Cultures were compared for monolayer-formation time, support of viral replication, passages and postinfection time required for expression of cytopathic effect (CPE), type of CPE, and virus yield. CPE was observed on the first passage with infected egg yolk in primary chicken embryo kidney cells, primary through tertiary chicken embryo liver (CEL) cells, and African green monkey kidney (VERO) cells; on the third blind passage of infected supernatant in Georgia bovine kidney cells, Crandall feline kidney cells, and baby hamster cells; on the fifth blind passage in rabbit kidney cells; and on the tenth blind passage in porcine kidney cells. CPE was not observed after 10 viral passages in rabbit bone-marrow cells. Monolayer formation time and postinfection time for CPE expression occurred sooner, and virus yield was greater, with CEL and VERO cells than with other cell lines.

Enhanced incidence of leg abnormalities in reovirus WVU 2937-infected chickens fed various dietary levels of selected vitamins.

Five experiments were conducted to evaluate the incidence of leg abnormalities of 4-, 6-, and 8-week-old reovirus WVU 2937-infected and uninfected chickens fed diets containing 20%, 100%, or 200% of the 1977 National Research Council nutrient requirements (NRC-77) of poultry for manganese, biotin, niacin, choline, or folic acid. Reovirus infection significantly elevated the incidence of leg abnormalities in the biotin-, niacin-, and folic-acid-deficient chickens and decreased the incidence of leg abnormalities in choline-deficient chickens. Reovirus-infected male chickens had a significantly greater incidence of leg abnormalities than reovirus-infected female chickens in the biotin, niacin, and folic acid studies. No sex differences in the incidence of leg abnormalities were observed in uninfected chickens. The incidence of leg abnormalities decreased with increasing dietary levels of biotin, niacin, choline, or folic acid in all chickens except reovirus-infected male chickens in the biotin study. In the manganese study, neither reovirus infection nor dietary level had any effect on the incidence of leg abnormalities in male or female chickens.

Responses of specific-pathogen-free chicks to concomitant infections of reovirus (WVU-2937) and infectious bursal disease virus.

Specific-pathogen-free white leghorn chicks concomitantly infected with both infectious bursal disease virus (IBDV) and reovirus (WVU-2937) on day 1 had significantly lower (P less than 0.05) virus-neutralizing- and precipitating-antibody geometric mean titers (GMT) to reovirus than chicks infected with only reovirus on day 1 but had a similar incidence of inflammation of the metatarsal digital flexor tendons. Chicks infected with IBDV on day 7 and reovirus on day 14 had a greater incidence of inflammation of the tarsometatarsal digital flexor tendons and lower neutralizing- and precipitating-antibody GMT to reovirus than chicks infected with only reovirus on day 14. Chicks infected with both viruses on day 1 had a significantly lower (P less than 0.05) neutralizing-antibody GMT to IBDV than chicks infected with only IBDV.