The application of glucose oxidase-labeled antibodies for the detection of proteins on nitrocellulose

We have evaluated the sensitivity of immunostaining with glucose oxidase for the detection of monomeric human serum albumin (HSA) and monomeric human immunoglobulin G (IgG). A modification of a histochemical procedure was utilized by replacing phenazine methosulfate (PMS) with 1-methoxyphenazine methosulfate (mPMS) and by replacing Tris-HCl with Tris-citrate to improve the solubility of the tetrazolium compounds tested. mPMS is less sensitive to light, and may be stored for long periods in solution; it is now used routinely by histochemists in place of PMS in dehydrogenase cytochemistry. pH values of 6.3-8.3 were tested, with the reaction at pH 8.3 providing a slight increase in sensitivity. The reaction rate increased markedly as the pH became more alkaline. The minimum quantity of HSA detected was 3 ng applied directly to nitrocellulose and 10 ng when blotted. Human IgG was routinely detected at 250 pg and occasionally at 100 pg when dotted on the nitrocellulose.     

C1q binding substances in pemphigus and bullous pemphigoid. Detection with a [131I] C1q binding assay.

A modification of the [125I]C1q binding assay was developed to allow the estimation of C1q binding activity (C1q BA) in pemphigus and bullous pemphigoid sera. The modifications include lower final concentration of PEG 6000 (1-5%) which permitted the use of sera that had been stored at -20 degrees C for extended periods of time; use of 131I instead of 125I and an [131I] C1q concentration of 5 microng/ml rather than 1 microng/ml. EDTA was used at a final concentration of 0-13 M to obviate the need for heat inactivation of sera. Sera from seventy-one patients with pemphigus and from 142 patients with bullous pemphigoid were tested for C1q BA. Of these 40% of the pemphigus and 20% of the bullous pemphigoid patients showed elevated C1q BA. A relationship between elevated C1q BA in serum and active disease was noted. Sequential samples from forty patients with pemphigus and thirty-seven patients with bullous pemphigoid demonstrated two different types of relationship between serum antibody titres to cutaneous antigens and C1a BA. In some patients serum antibody titres and C1q BA increased and decreased simultaneously; in others, increase of C1q BA followed increase of antibody titre and coincided with its decrease. The latter relationship supports the hypothesis that C1q BA may represent at least in part antigen-antibody complexes containing cutaneous antigens.     

Serum and blister fluid immune complexes in bullous pemphigoid: detection with C1q and monoclonal rheumatoid factor.

Eighty serum samples and 24 blister fluids from 51 patients with active bullous pemphigoid were tested for the presence of immune complexes by both a monoclonal rheumatoid factor (mRF) inhibition radioassay and a C1q-binding radioassay. Forty-two of the 80 serum samples were positive by the mRF assay, while 27 were positive by the C1q-binding assay. Antibody titres to the basement membrane zone did not correlate with levels of circulating immune complexes. Thirteen of 24 blister fluids had detectable immune complexes by the C1q assay, while only seven of 24 blister fluids were positive by the mRF assay. Sucrose density-gradient ultracentrifugation studies suggest that the mRF- and C1q-reactive substances in both bullous pemphigoid sera and blister fluids are of a size compatible with immune complexes. Although immune complexes are detectable in a high percentage of bullous pemphigoid patients, their role in this disease may be epiphenomenal rather than pathogenetic, merely reflecting the presence of autoantibody and soluble antigen.     

Treatment of infants with ureteropelvic junction obstruction: findings from the PURSUIT network

International Urology and Nephrology - Studies based on administrative databases show that infant pyeloplasty is associated with minority race/ethnicity but lack clinical data that may influence...     

The Effect of Stress and Meditation on Salivary Protein and Bacteria: A Review and Pilot Study

Abstract

Dentally-induced stress and relaxation-induced anxiety reduction have been correlated with salivary changes in dental patients in two recent studies. In two subsequent studies, test anxiety-induced stress and relaxation-induced anxiety reduction were correlated with salivary changes in dental students. In another study using the resazurin dye indicator, increased salivary bacterial levels were correlated with an increased dental caries incidence. As a result of these findings, it was decided to reinvestigate the effects of stress and relaxation on salivary changes and in addition to examine the effects of those conditions on salivary bacteria. The hypotheses under consideration were: (1) Salivary changes from stress to relaxation will be from opaque to translucent and from high to low protein levels; and (2) salivary bacteria will increase under the condition of stress and decrease under the condition of relaxation. The subjects were twelve dental students. Stress and relaxation were evaluated before and after meditation by verbal reports and examination of saliva for opacity, translucency, protein and bacteria (resazurin dye method). There were significant anxiety-reduction changes by the end of the meditation sessions (p < 0.001) as measured by increased salivary translucency, decreased salivary protein and reduced subjective evaluation of stress. Using the resazurin dye method, bacterial levels showed a significant decrease by the end of the meditation sessions (p < 0.001). The results support hypothesis 1 and reaffirm previous findings in regard to the effectiveness of: (1) salivary changes as measures of stress and relaxation; and (2) meditation to induce deep relaxation. The finding of high bacteria levels under stress and lower bacterial levels under relaxation supports hypothesis 2 and indicates that stress may contribute to dental caries and relaxation may have an anti-caries effect.     

Vitamin B12-dependent biosynthesis ties amplified 2-methylhopanoid production during oceanic anoxic events to nitrification

Bacterial hopanoid lipids are ubiquitous in the geologic record and serve as biomarkers for reconstructing Earth’s climatic and biogeochemical evolution. Specifically, the abundance of 2-methylhopanoids deposited during Mesozoic ocean anoxic events (OAEs) and other intervals has been interpreted to reflect proliferation of nitrogen-fixing marine cyanobacteria. However, there currently is no conclusive evidence for 2-methylhopanoid production by extant marine cyanobacteria. As an alternative explanation, here we report 2-methylhopanoid production by bacteria of the genus Nitrobacter, cosmopolitan nitrite oxidizers that inhabit nutrient-rich freshwater, brackish, and marine environments. The model organism Nitrobacter vulgaris produced only trace amounts of 2-methylhopanoids when grown in minimal medium or with added methionine, the presumed biosynthetic methyl donor. Supplementation of cultures with cobalamin (vitamin B₁₂) increased nitrite oxidation rates and stimulated a 33-fold increase of 2-methylhopanoid abundance, indicating that the biosynthetic reaction mechanism is cobalamin dependent. Because Nitrobacter spp. cannot synthesize cobalamin, we postulate that they acquire it from organisms inhabiting a shared ecological niche—for example, ammonia-oxidizing archaea. We propose that during nutrient-rich conditions, cobalamin-based mutualism intensifies upper water column nitrification, thus promoting 2-methylhopanoid deposition. In contrast, anoxia underlying oligotrophic surface ocean conditions in restricted basins would prompt shoaling of anaerobic ammonium oxidation, leading to low observed 2-methylhopanoid abundances. The first scenario is consistent with hypotheses of enhanced nutrient loading during OAEs, while the second is consistent with the sedimentary record of Pliocene–Pleistocene Mediterranean sapropel events. We thus hypothesize that nitrogen cycling in the Pliocene–Pleistocene Mediterranean resembled modern, highly stratified basins, whereas no modern analog exists for OAEs.

Hopanoids are a structurally diverse class of isoprenoid lipids that are involved in bacterial membrane homeostasis by mediating membrane organization and stress response (1–4). As chemical fossils, hopanoids and their diagenetic products are ubiquitous in the geologic record where they serve as important biomarkers for our planet’s biogeochemical and microbial evolution from the Proterozoic onward (5–8). Specifically, a subgroup of hopanoids methylated at the C-2 position (2-methylhopanoids) has been used as biomarkers for cyanobacteria (9) and invoked as evidence for the proliferation of nitrogen-fixing cyanobacteria during intervals such as Mesozoic ocean anoxic events (OAEs) (10, 11) and the Paleocene–Eocene Thermal Maximum (12).The importance of hopanoids as biomarkers has sustained interest in understanding their sources and their role in bacterial physiology, leading to multiple recent studies that challenge prior assumptions (2, 3, 13–15). Specifically, the occurrence of 2-methylhopanoids in diverse alphaproteobacteria, including the anoxygenic phototroph Rhodopseudomonas palustris (16) and other freshwater and soil bacteria (17–21), illustrates that 2-methylhopanoids are not exclusive to cyanobacteria. Although 2-methylhopanoids predominantly originate from cyanobacteria in environments such as freshwater and lagoonal microbial mats (22, 23), it is plausible that other bacteria could have contributed to the geologic record of 2-methylhopanoids. However, it is unlikely that freshwater cyanobacteria were primary contributors to the accumulation of 2-methylhopanoids in offshore marine environments during Mesozoic OAEs. Thus, numerous uncertainties surround the origin of 2-methylhopanoids in the geological record and the reasons behind their prevalence during episodes of ocean anoxia.Screening of genomes and metagenomes for the hpnP gene encoding a hopanoid C-2 methyltransferase (24) has led to the identification of a small subset of freshwater and soil bacteria as putative 2-methylhopanoid producers but has not revealed instances in marine cyanobacteria (24, 25). A more recent gene homology analysis suggested the presence of the hpnP gene in diverse marine cyanobacteria (26). However, 2-methylhopanoids have not been detected in any of these cyanobacteria (15), suggesting this recent study (26) may have detected related genes of different function.Identification of common source organisms and elucidation of the underlying biochemistry of 2-methylhopanoid biosynthesis could help constrain the factors controlling their geologic record. Based on previous detection of the hpnP gene in a species of the alphaproteobacterial genus Nitrobacter (24), we hypothesized that Nitrobacter spp. could be an important but previously overlooked source of 2-methylhopanoids. Nitrobacter spp. are nitrite-oxidizing bacteria (NOB) that are abundant in soil, fresh water, and the oceans where they share an ecological niche with other NOB (Nitrospina, Nitrospira, Nitrococcus spp.), ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (27–29).Here, we use a combination of genomic analyses and culture experiments with the model organism Nitrobacter vulgaris AB1 to elucidate the factors driving 2-methylhopanoid biosynthesis in this taxon. Our results suggest that the reaction mechanism is not only dependent on a radical S-adenosylmethionine (SAM) enzyme (23) but also on the enzymatic cofactor cobalamin (vitamin B₁₂). Because Nitrobacter spp. are cobalamin auxotrophs, we hypothesize that synergistic interaction between Nitrobacter and cobalamin-producing nitrifying archaea could be an important control on 2-methylhopanoid production. This hypothesis is consistent with enhanced production of 2-methylhopanoids under conditions of intensified oxidative nitrogen cycling in past environments.     

The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase

Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2^−/− mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2^−/− mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2^−/− brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.Determining the genetic basis for rare hereditary human diseases has benefited from advances in DNA sequencing technologies (1). As a greater number of disease-causing mutations are mapped, however, it is also becoming apparent that many of the affected genes code for poorly characterized proteins. Assigning biochemical and cellular functions to these proteins is critical to achieve a deeper mechanistic understanding of human genetic disorders and for identifying potential treatment strategies.Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurologic syndrome marked by spasticity and lower extremity weakness (2). Many genetic types of HSP have been identified and are numbered according to their order of discovery [spastic paraplegia (SPG) 1-72] (2, 3). Of these genetic variants, more than 40 have been mapped to causative mutations in protein-coding genes. HSP genes code for a wide range of proteins that do not conform to a single sequence- or function-related class. A subset of HSP genes, including PNPLA6 (or neuropathy-target esterase) (SPG39) (4), DDHD1 (SPG28) (5), and DDHD2 (SPG54) (3, 6–8), code for serine hydrolases. These enzymes have been designated as (lyso)phospholipases based on in vitro substrate assays (9–11), but their endogenous substrates and physiological functions remain poorly understood. The mutational landscape that affects these lipid hydrolases to cause recessive HSP is complex but collectively represents a mix of null and putatively null and/or functional mutations. Moreover, the type of HSP appears to differ in each case, with DDHD1 mutations causing uncomplicated HSP, whereas PNPLA6 and DDHD2 mutations lead to complex forms of the disease that exhibit additional phenotypes including, in the case of DDHD2, intellectual disability. Human subjects with DDHD2 mutations also displayed evidence of brain lipid accumulation as detected by cerebral magnetic resonance spectroscopy (6). Both rodent and human DDHD2 enzymes are highly expressed in the brain compared with most peripheral tissues (6, 9); however, the specific lipids regulated by DDHD2 in the central nervous system (CNS) have not yet been identified.Determining the metabolic function of DDHD2 in the brain is an important step toward understanding how mutations in this enzyme promote complex HSP and for identifying possible therapeutic strategies for the disease. Toward this end, we report herein the generation and characterization of DDHD2^−/− mice and a selective DDHD2 inhibitor. DDHD2^−/− mice exhibit defects in movement and cognitive function. Mass spectrometry (MS)-based lipidomics (12, 13) revealed a striking and selective elevation in triglycerides (triacylglycerols, or TAGs) throughout the CNS, but not in peripheral tissues, of DDHD2^−/− mice. This metabolic change correlated with pervasive lipid droplet (LD) accumulation in neuronal cell bodies of DDHD2^−/− mice. Biochemical assays confirmed that DDHD2 possesses TAG hydrolase activity. Finally, wild-type mice treated subchronically with a DDHD2 inhibitor also exhibited significant elevations in CNS TAGs. These data, taken together, indicate that DDHD2 is a principal TAG hydrolase of the mammalian brain and point to deregulation of this pathway as a major contributory factor to complex HSP.     

Spinal manipulation does not affect pressure pain thresholds in the absence of neuromodulators: a randomized controlled trial

Background: Measurement of pressure pain threshold (PPT) is a way to determine one of the many potential treatment effects of spinal manipulative therapy.

Objective: To determine how multiple spinal manipulations administered in a single-session affected PPTs at local and distal sites in asymptomatic individuals.

Methods: Participants were randomly assigned into one of three groups: Group one (n = 18) received a lumbar manipulation followed by a cervical manipulation. Group two (n = 17) received a cervical manipulation followed by a lumbar manipulation. The control group (n = 19) received two bouts of five minutes of rest. At baseline and after each intervention or rest period, each participant’s PPTs were obtained using a handheld algometer. The PPTs were tested bilaterally over the lateral epicondyles of the humerus and over the mid-bellies of the upper trapezius, lumbar paraspinal, and the tibialis anterior muscles. This study was registered with ClinicalTrials.gov, and its Identifier is NCT02828501.

Results: Repeated-measures ANOVAs and Kruskal–Wallis tests showed no significant within- or between-group differences in PPT. Within-group effect sizes in the changes of PPT ranged from ?.48 at the left paraspinal muscles to .24 at the left lateral humeral epicondyle. Statistical power to detect significant differences at α of 0.05 was calculated to be 0.94.

Conclusions: This study suggests that in young adults who do not have current or recent symptoms of spinal pain, multiple within-session treatments of cervical and lumbar spinal manipulation fail to influence PPTs. Changes in PPT that are observed in symptomatic individuals are likely to be primarily influenced by pain-related neuromodulators rather than by an isolated, mechanical effect of spinal manipulation.