Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of proteins in human cerebrospinal fluid

Matrix-assisted laser desorption/ionization time-of-flight mass spectra of proteins in cerebrospinal fluid analyzed without prior purification are presented. Less than 100 fmol amounts of proteins in the 10,000 to 20,000 u mass range and linked to human disease (multiple sclerosis, Alzheimer's disease, and stroke) were detected in a complex mixture of proteins and peptides, in the presence of high concentrations of salts, lipids and free amino acids. The mass resolution was sufficient to distinguish between the non-hydroxylated and hydroxylated forms of a 13,400 u protein. Simple fractionation of the cerebrospinal fluid using microbore-reversed phase high performance liquid chromatography improved signal-to-noise ratios in the mass spectra. High-accuracy peptide mass mapping and database searching were utilized to confirm the identity of several proteins. The presented results show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry could be used as a tool to perform rapid screening of chemically altered proteins in small volumes of biological fluids.     

Localization of the ROMK potassium channel to the apical membrane of distal nephron in rat kidney

BACKGROUND: The apical potassium (K+) channels mediate K+ recycling in thick ascending limb (TAL) and K+ secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K+ channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K+ channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. METHODS: Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. RESULTS: The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a approximately 42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H+-ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. CONCLUSIONS: These results further strengthen the idea that ROMK channels play important roles in the recycling of K+ in TAL and the secretion of K+ in CCD.     

Clinical trials and treatment effects monitoring

Treatment effects monitoring is the act of reviewing accumulated data by treatment group to determine whether a trial should continue unaltered. That monitoring is required for any trial where there is a risk of an aggregate form of harm for subjects because of continued use of an inferior treatment or because of failure to use a superior treatment. Institutional review boards (IRBs), in deciding whether to approve such research, require that (when appropriate) there is adequate provision for monitoring the data collected to ensure the safety of subjects. The focus here is on the conditions necessary to satisfy this requirement. The conditions discussed are timeliness of the monitoring, completeness of data for monitoring, competency of monitors, and freedom of monitors to act and recommend as they deem necessary, regardless of the wishes, desires, or dictates of sponsors.     

Intraarticular foot and ankle injections to identify source of pain before arthrodesis

OBJECTIVE: The purpose of our study was to evaluate the usefulness of diagnostic joint injections in patients with foot and ankle pain when the radiologist attempts to identify the source of pain. This study also correlated the results of injection with outcome after arthrodesis. MATERIAL AND METHODS: We retrospectively reviewed the records of 22 patients who had a foot or ankle joint injected to identify a source of pain and who later underwent arthrodesis of the painful joint. All patients had long-term foot and ankle symptoms of variable causes. Twenty-four joints were assessed: 13 subtalar, five talonavicular, four ankle, one calcaneocuboid, and one metatarsocuneiform. All patients had plain radiographs, 11 had CT studies, and five had bone scans. Contrast material was used to assess adequate positioning of the needle inside the joint before injection. All joints were injected under fluoroscopic control. Steroid was added in eight joints. After injection, patients were assessed for relief of symptoms. Patients subsequently underwent arthrodesis on the basis of the results of the injection. RESULTS: In 20 patients (22 joints), long-term follow-up showed that injections allowed us to correctly identify the source of pain and successfully guide arthrodesis. Of these 20 patients, 17 had significant pain relief after injection and fusion, whereas three patients had mild or no response. With one of these patients, we injected other joints and changed surgical plans. One of the two remaining patients had more pain relief after injection than after arthrodesis. The other patient had no relief after injection, but subsequent fusion because of persistent pain was successful. We found imaging studies to be less useful than diagnostic injections when we were attempting to identify the source of pain. CONCLUSION: Intraarticular injection of anesthetic in painful foot and ankle joints helped us confirm the source of pain in 20 of 22 patients, which in turn led to successful arthrodesis and good outcomes for these patients.     

The reinforced laryngeal mask airway (LMA) as an alternative airway device to manage the difficult airway

BACKGROUND: Infection with human parvovirus B19 (B19) has been reported in a few patients with various vasculitis syndromes. Mixed cryoglobulinaemia (MC), a model of small vessel size vasculitis, may result from numerous infectious diseases, particularly hepatitis C virus (HCV) infection. AIM: To assess the prevalence of seric B19 infection markers in a large series of patients with MC, with or without HCV infection. PATIENTS AND METHODS: Sixty-four patients were studied: essential MC (EMC, n = 19), MC associated with non-infectious diseases (non-essential MC, n = 9), and patients with HCV infection with (HCV-MC, n = 18) or without MC (HCV-no-MC, n = 18). Patients were considered to have MC if two successive determinations of their serum cryoglobulin concentration were above 0.05 g/l. Serum samples were analysed for specific IgG and IgM antibodies to B19 by enzyme immunoassay. B19 DNA detection was performed by polymerase chain reaction using a set of primers located in the VP1 gene, separately in serum and in cryoprecipitates to investigate a possible capture of B19 DNA in cryoprecipitate. The study also looked for a possible enrichment for of IgG antibodies to B19 in MC. RESULTS: The presence of specific IgG antibodies to B19 was found in 68% EMC, 56% non-essential MC, 78% HCV-MC, and 78% HCV-no-MC. No patient of either group had specific IgM antibodies to B19, or B19 DNA in serum or in cryoprecipitate. Overall, IgG antibodies to B19 were found in 46 of 64 (72%) serum samples, a prevalence quite similar to the prevalence in general adult population (> 60%). A specific enrichment of IgG antibodies to B19 in the MC was not found. CONCLUSION: These results suggest that B19 infection is neither an aetiological factor of EMC, nor a cofactor that may lead to MC production in patients with chronic HCV infection.     

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: a problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.     

Simplified technique for detection of significant bacteriuria by microscopic examination of urine

A comparative study of microscopic examination of 10 microl (simplified loop technique) and 50 microl (traditional drop technique) of uncentrifuged Gram-stained urine specimens for detection of significant bacteriuria was carried out. The results demonstrated that the 10-microl loop technique can be used as an alternative to the 50-microl drop technique for presumptive diagnosis of urinary-tract infection in bacteriological practice, with the advantages of greater rapidity and ease of performance.     

Temporary vascular occlusion during cerebral aneurysm surgery

Mood disorders and attention deficit-hyperactivity disorder (ADHD) co-occur in 20-30% of children and adolescents diagnosed in both epidemiological and clinical studies, but little information is available regarding cognitive factors that may be relevant to the expression of co-occurring mood disorders and ADHD. This study examined whether ADHD with and without a comorbid mood disorder could be differentiated on the basis of cognitive factors associated with prominent theories of depression. Children meeting diagnostic criteria for ADHD (n = 14) or ADHD and a comorbid mood disorder (n = 27) were assessed on a variety of cognitive indices. Children in the comorbid group reported more negative views of themselves and a more depressogenic attributional style. Cognitive disturbances associated with A. T. Beck's (1967) cognitive model and attributional style theories of depression differentiate ADHD children with significant mood pathology.     

Direction determination in the minus-end-directed kinesin motor ncd

Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.