Development and testing of a field diagnostic assay for peste des petits ruminants virus.

We have developed an immunochromatographic test for the diagnosis of peste des petits ruminants (PPR) under field conditions. The diagnostic assay has been tested in the laboratory and also under field conditions in Ivory Coast, Pakistan, Ethiopia and Uganda. The test is carried out on a superficial swab sample (ocular or nasal) and showed a sensitivity of 84% relative to PCR. The specificity was 95% over all nasal and ocular samples. The test detected as little as 10(3) TCID50 (50% tissue culture infectious doses) of cell culture-grown virus, and detected virus isolates representing all four known genetic lineages of peste des petits ruminants virus. Virus could be detected in swabs from animals as early as 4 days post-infection, at a time when clinical signs were minimal. Feedback from field trials was uniformly positive, suggesting that this diagnostic tool may be useful for current efforts to control the spread of PPR.

From chromosomal alterations to target genes for therapy: integrating cytogenetic and functional genomic views of the breast cancer genome.

A vast number of recurrent chromosomal alterations have been implicated in cancer development and progression. However, most of the genes involved in recurrent chromosomal alterations in solid tumors remain unknown, despite the recent substantial progress in genomic research and availability of high-throughput technologies. For example, it is now possible to quickly identify large numbers of differentially expressed genes in cancer specimens using cDNA microarrays. Integration of this "functional genomic view" of the cancer genome with the "cytogenetic view" could lead to the identification of genes playing a critical role in cancer development and progression. In this review, we illustrate how the combination of three different microarray technologies, cDNA, CGH, and tissue microarrays, makes it possible to directly identify genes involved in chromosomal rearrangements in cell line model systems and then rapidly explore their significance as potential diagnostic and therapeutic targets in human primary breast cancer progression.

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum.

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.

Characterization of a thermostable DNA glycosylase specific for U/G and T/G mismatches from the hyperthermophilic archaeon Pyrobaculum aerophilum.

U/G and T/G mismatches commonly occur due to spontaneous deamination of cytosine and 5-methylcytosine in double-stranded DNA. This mutagenic effect is particularly strong for extreme thermophiles, since the spontaneous deamination reaction is much enhanced at high temperature. Previously, a U/G and T/G mismatch-specific glycosylase (Mth-MIG) was found on a cryptic plasmid of the archaeon Methanobacterium thermoautotrophicum, a thermophile with an optimal growth temperature of 65 degrees C. We report characterization of a putative DNA glycosylase from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The open reading frame was first identified through a genome sequencing project in our laboratory. The predicted product of 230 amino acids shares significant sequence homology to [4Fe-4S]-containing Nth/MutY DNA glycosylases. The histidine-tagged recombinant protein was expressed in Escherichia coli and purified. It is thermostable and displays DNA glycosylase activities specific to U/G and T/G mismatches with an uncoupled AP lyase activity. It also processes U/7,8-dihydro-oxoguanine and T/7,8-dihydro-oxoguanine mismatches. We designate it Pa-MIG. Using sequence comparisons among complete bacterial and archaeal genomes, we have uncovered a putative MIG protein from another hyperthermophilic archaeon, Aeropyrum pernix. The unique conserved amino acid motifs of MIG proteins are proposed to distinguish MIG proteins from the closely related Nth/MutY DNA glycosylases.

A urinary marker for occult systemic coccal disease.

Gram-negative particles, found in 'normal' urine by an improved microscopic technique, become excessive (often > 10(5)/ml) in many systemic diseases. In these diseases they are accompanied by sparse, usually fastidious, gram-positive cocci. Antibiotics at moderate doses usually have little effect. Larger doses of antibiotics suppress, or temporarily eliminate, the particles. In this report, the particles are characterized by light microscopy for better identification. Then, by detection of muramic acid in a hydrolysate and by transmission electron microscopy, they are identified as decomposed ('exploded') gram-positive cocci. Since explodeds cannot be external contaminants, and their precursors cannot proliferate sufficiently in urine, they must have crossed renal membranes to come from within the body. They are demonstrated in tissue fluids and in synovial fluids by optical microscopy, by their muramic acid, and by transmission electron microscopy. Thus, they cross other membranes. Explodeds are excessive in several rheumatic diseases, in renal diseases, in diseases in which a coccal cause has been sought, and in some in which cocci have never been considered. There is no precedent for explodeds. Their appearance and numbers are compatible with the literature on natural and experimental systemic streptococcal diseases and with the experimental illnesses following injection of streptococcal cell walls. Urinary explodeds are likely to be the end result of the 'almost physiological' entry of streptococci into the circulation which necessitates predental antibiotic prophylaxis in mitral disease. Increased numbers of urinary explodeds probably represent excessive entry of precursors or proliferation of precursors within the host. Urinary explodeds serve as a marker for diverse systemic diseases, systemic coccal disease.

Computer algorithm offers a comprehensive view of quantitative bacteriuria.

Using improved microscopy of urine sediment, the consistent finding of bacteria, usually gram-positive cocci at low counts, free or even in casts, in the sediment of carefully collected urines from patients with systemic illnesses has led to the need to reconsider their exclusion by the customary criteria for 'significance' of bacteria in urine. Since 'significance' is currently based upon mathematical assumptions limited to high counts (> 10(5) colony-forming units/ml) for the prediction of clinical pyelonephritis alone, a digital computer program was created to predict the full spectrum of the expected concentration of bacteria in bladder urine versus time for a very wide range of possible bacterial division times, bladder kinetics and urine flow rates. Curves generated resulted in the discovery of very simple rules based on an easily calculated discriminant, the host's critical division time (CDT) for any bacterial species in his urine. (1) If the division time in the urine of a species entering the urinary tract is shorter than the CDT, the bacteria will proliferate to > 10(5) cfu/ml. Published data on growth in human urine show that very few bacterial species can divide so fast in urine, and those are the ones currently considered 'significant'. Except for some enterococci, streptococci cannot. (2) With a division time only marginally longer than the CDT, any bacterium would wash out unless continuously supplemented via the kidney or from the bladder wall. (3) With a continued supplement and the longer division time, the concentration would fall to a low plateau, and that plateau is diagnostic of a continued supplement. The cocci observed by microscopy are fastidious or dead. They grow poorly if at all in urine, and thus are not likely to ascend the urinary tract. Their appearance corresponds to the earlier studies of bacteriuria and to the known excretion of blood-borne bacteria in natural disease, whether or not there are anatomical changes in the kidney. It is suggested that the low-level coccal bacteriuria found is a marker for scent bacteremia in many systemic diseases for which a bacterial provocation has been sought.

Preparation of plasmid DNA by sequential enzymatic digestion.

A new method for the preparation of plasmid DNA from Escherichia coli, sequential enzymatic digestion, is described. The method is based on sequential and selective enzymatic digestion of all components of E. coli except for the supercoiled plasmid DNA. The key enzymes are exonuclease I and exonuclease III that specifically hydrolyze linear chromosomal DNA and are unable to attack supercoiled plasmid DNA under controlled conditions. Isolated plasmid DNA can be sequenced and digested with restriction enzymes.

Improved microscopic detection of bacteriuria.

A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as "significant" by the conventional laboratory culture. More importantly, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.

A new method of sequencing DNA.

An entirely new method of sequencing DNA has been devised that does not use electrophoresis, radioactivity, or fluorescence. The method works by measuring pyrophosphate generated by the DNA polymerization reaction. DNA and DNA polymerase are held by a DEAE-Sepharose column and solutions containing different dNTPs are pumped through. The pyrophosphate generated is measured continuously by a device consisting of a series of columns containing enzymes covalently attached to Sepharose. The alternating copolymer poly(dA.dT) is sequenced as an illustration of the method. Future improvements that will facilitate automation are discussed.

Acquired iron-deficiency anemia caused by an antibody against the transferrin receptor.

We report a case of anemia due to autoantibodies to the transferrin receptor interfering with iron incorporation by erythroid progenitors. A previously healthy woman with severe acquired microcytic anemia had increased serum iron levels, electrophoretically normal transferrin concentrations, and very high levels of free protoporphyrin in red cells. The bone marrow had no stainable iron but had an excess of normal-appearing plasma cells. Erythroid precursors stained with fluorescent mouse antihuman IgM. The serum contained an antibody that reduced 59Fe incorporation by erythroleukemia K562 cells in vitro but did not inhibit iron transferrin binding. An IgM fraction of the patient's serum immunoprecipitated the human transferrin receptor obtained from solubilized [35S]methionine-labeled K562 membranes. Binding of [59Fe]transferrin or fluorescent iron transferrin was not diminished by the patient's serum at 4 degrees C, but at 37 degrees C uptake was markedly reduced, as was the binding of fluorescent monoclonal antibodies to either surface transferrin or the human transferrin receptor. A complete clinical and hematologic remission occurred with azathioprine and prednisone therapy. We conclude that the patient's autoreactive IgM down-regulated the number of transferrin receptors and diminished iron incorporation by erythroblasts, leading to an iron-deficiency anemia.

Acquired iron-deficiency anaemia due to impaired iron transport.

A previously healthy 39-year-old woman presented with severe iron-deficiency anaemia, but she had lost no blood and her serum iron level was high. Her bone marrow was hypercellular with a predominance of erythroid elements and had no stainable iron deposits, but it also showed dyserythropoiesis and an excess of apparently normal plasma cells. IgM was demonstrated on her bone-marrow erythrocytes and their precursors. On azathioprine and prednisone therapy she had a complete clinical and haematological remission. The impaired iron transport and the associated dyserythropoiesis were probably due to an IgM-mediated autoimmune process. Diabetes mellitus, which first appeared during her anaemic illness, could also have been due to an autoimmune process. This is the first report of an iron-deficiency anaemia caused by a naturally acquired impairment of iron transport.

Acute appendicitis in early childhood.

1728 appendectomies were performed in our institution between the years 1973 and 1978, only 18 of them below the age of 5 years. Acute appendicitis in this age is characterized by a very low incidence (1%); an overwhelming male predominance (8:1), a short history, and a rapid progress of the disease. In 72% perforation of appendix and peritonitis were present at operation. The triad of fever, abdominal pains and vomiting was present in all cases. A high leucocyte count was noted in all cases except one. All patients were operated upon within 16 h of admission. No mortality, a low morbidity and a short hospital stay were recorded.

Electromagnetic field scattering by a triangular aperture.

The multiple Laplace transform has been applied to analysis and computation of scattering by a double triangular aperture. Results are obtained which match far-field intensity distributions observed in experiments. Arbitrary polarization components, as well as in-phase and quadrature-phase components, may be determined, in the transform domain, as a continuous function of distance from near to far-field for any orientation, aperture, and transformable waveform. Numerical results are obtained by application of numerical multiple inversions of the fully transformed solution.

Electrogenesis from an ATPase-ATP-sodium pseudo pump.

The short circuit current and the open circuit voltage responses of membranes to ATP, which have been attributed to membrane ATPase acting as a sodium pump, have been reproduced not only in a lipid membrane containing solubilized ATPase but also in membranes formed of the phospholipids contained in ATPase. The response is greatest with cardiolipin, but occurs with other acidic phospholipids. This observation of electrogenesis without hydrolysis is a surface phenomenon probably due to the alignment of ATP on the phospholipid by ion association at its interface with the water phase. The finding constitutes a precaution for interpreting studies of membrane Na-K-ATPase or for its incorporation into an artificial membrane. The substances necessary for electrogenesis are present at the mitochondrial membrane, and the particular orientation of the ATP on the phospholipids in vitro suggests a role for this ion association in the function of Na-K-ATPase.