comB, a novel competence gene required for natural transformation of Acinetobacter sp. BD413: identification, characterization, and analysis of growth-phase-dependent regulation.

Here we describe five tandemly arranged and converging ORFs in Acinetobacter sp. BD413, namely lytB, orfY, orfX, comB, and orfZ, located upstream of the previously identified competence gene comC. The N-termini of the deduced proteins OrfY and ComB exhibit the conserved endopeptidase cleavage motifs of prepilin proteins; the deduced protein ComB is similar to type IV pilins. LytB is similar to the Escherichia coli LytB, which has been implicated in the stringent response. No homologues of OrfX, OrfY and OrfZ could be identified. A mutation in orfY or orfZ led to 100-fold reduced transformation frequencies and a mutation in comB resulted in a non-competent phenotype. Disruption of lytB did not affect the natural transformation phenotype. Complementation studies clearly demonstrated that comB is involved in natural transformation, whereas the transformation-deficient phenotypes of orfY and orfZ mutants were due to polar effects on comB and comC, respectively. Analyses of the twitching motility phenotype and of the ultrastructure of the noncompetent comB mutant suggested that the competence gene comB is not essential for the biogenesis of type IV pili and expression of the type IV pili-associated property of twitching motility. Transcriptional fusions between comB and a promoter-free lacZ gene were constructed, and analysis of growth-phase-dependent transcription revealed increased expression of comB during prolonged exponential and stationary phases.

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli.

The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.

[Liver tumors in children: spiral CT findings and differential diagnostic classification]. / Lebertumoren bei Kindern: Spiral-CT-Befunde und ihre differentialdiagnostische Einordnung.

Liver tumors in childhood are rare. They can be subdivided in benign or primary and secondary malignant liver tumors. The prognosis of malignant liver disease has improved due to extended therapy in the past few years. Thus, diagnosis of liver tumors in childhood gains in significance. After a suspect ultrasound diagnosis, usually computed tomography (CT) is used as a radiologic sectioning method. Guided by a retrospective analysis of 15 cases, the importance of the Helical-CT is presented. Helical-CT is a good diagnostic method for pediatric cases due to reduced scan times. By the basis of clinical data, distribution, and density, various differential diagnoses can be made. Benign liver tumors, especially cysts and frequently haemangioma, can easily be diagnosed and controlled because of their typical echogenicity and contrast visualisation. In cases of hamartoma, focal nodal hyperplasia, and adenoma computed tomography diagnosis is often necessary. Primary malignant liver tumors can prove to be unilocular hepatoblastomas or hepatocellular carcinomas, while disseminated liver invasions usually are considered as metastatic liver tumors of kidney or adrenal cancer.

[Bactericidal permeability increasing protein (BPI-ANCA marked chronic inflammatory bowel diseases and hepatobiliary diseases]. / Bactericidal Permeability Increasing Protein (BPI)-ANCA markiert chronische entzündliche Darmerkrankungen und entzündliche hepatobiliäre Erkrankungen.

BACKGROUND: Bactericidal permeability increasing protein (BPI) is an antibacterial product of neutrophilic granulocytes that can serve as target antigen for antineutrophil cytoplasmic antibodies (ANCA). The clinical associations of autoantibodies against BPI (BPI-ANCA) are essentially unclear. PATIENTS AND METHODS: 587 sera from patients with chronic inflammatory bowel diseases, inflammatory hepatobiliary diseases, primary systemic vasculitides and other rheumatological diseases were examined for BPI-ANCA by mono-specific ELISA and a standard indirect immunofluorescence test for ANCA. (ACD-CPR versus S-CPR). The treatment groups were similar with respect to age, sex, time interval from collapse to CPR, defibrillation and first epinephrine medication. There was no difference between the ACD group and the standard CPR group in terms of survival rates and neurologic outcome. No differences occurred concerning complications of CPR. CONCLUSION: In our two-tiered EMS system with physician-staffed ambulances ACD-CPR neither improved nor impaired the survival rates and the neurological prognosis in patients with out-of-hospital cardiac arrest. Our results are in accordance with other studies carried out in EMS systems, with first tier call-response intervals between 4 and 6 min. RESULTS: The prevalence of BPI-ANCA was 43% in ulcerative colitis, 23% in Crohn's disease, 35% in primary sclerosing cholangitis, 25% in primary biliary cirrhosis and 29% in autoimmune hepatitides. In a spectrum of systemic vasculitides, inflammatory joint diseases and collagen vascular diseases the prevalence was only 3 to 11%. In contrast to PR3-ANCA and MPO-ANCA, BPI-ANCA was not associated with a particular pattern of fluorescence in the immuno-fluorescence test on ethanol- and formalin-fixed neutrophils. CONCLUSION: This study shows that BPI-ANCA is the third ANCA specificity, besides PR3-ANCA and MPO-ANCA, with a limited spectrum of clinical associations. The diagnostic and prognostic relevance of BPI-ANCA in the above clinical conditions is being examined prospectively.

Anti-neutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability increasing protein (BPI): a new seromarker for inflammatory bowel disease and associated disorders.

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to be associated with ANCA, such as ANCA-associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary sclerosing cholangitis. The immunoreactivity of recombinant PBI (rBPI) was established in order to develop an ELISA specific for rBPI. By means of this assay, BPI-ANCA were assessed in sera of 178 patients with IBD or the associated disorder primary sclerosing cholangitis, 112 patients with ANCA-associated vasculitides, and in sera of 182 disease and 140 health controls. BPI-ANCA were found to be closely associated with IBD and primary sclerosing cholangitis (34% and 44% of ANCA-positive sera, respectively). By contrast, BPI-ANCA positivity was low (<10%) in the double-negative sera of patients with ANCA-associated vasculitides and in disease and health controls. BPI-ANCA appear to constitute an important marker for IBD and primary sclerosing cholangitis, but not for the ANCA-associated vasculitides.

Impaired sensitivity to visual contrast in children treated early and continuously for phenylketonuria.

Contrast sensitivity was assessed in 47 children aged 5.4-9.8 years: 12 with phenylketonuria (PKU), six unaffected siblings and 29 children from the general population. Children with PKU, despite early and continuous treatment and despite phenylalanine (Phe) levels within accepted limits, were impaired across the range of spatial frequencies [1.5-18.0 cycles per degree of visual angle (c.p.d.)]. They were most impaired at the next to the highest spatial frequency, where "group' accounted for 70% of the variance in sensitivity to contrast, controlling for acuity, sex, age and test site. Never, at any spatial frequency, was the contrast sensitivity of any PKU subject better than that of his or her sibling. All subjects were tested under conditions of 20/20 vision, with correction if needed. The mean IQ of PKU subjects was 99; IQ was not significantly related to contrast sensitivity performance. We interpret these findings as support for Diamond's hypothesis that moderately elevated plasma Phe levels (3-5 x normal), combined with reduced plasma tyrosine (Tyr), moderately reduce the levels of Tyr reaching the eye and brain, which adversely affects those dopamine neurons that fire and turn over dopamine most rapidly (the dopamine neurons in the retina and those projecting to prefrontal cortex). This would lead to the deficit in contrast sensitivity found here and to the selective deficit in prefrontal cortex cognitive functions previously reported in PKU children under moderately good dietary control.

Prevalence of vision and ocular disease conditions in a clinical pediatric population.

BACKGROUND: The purpose of this study was to fill a significant void in the ophthalmic literature by performing a large scale, comprehensive, prospective study of the prevalence of vision disorders and ocular pathology in a clinical pediatric population using well-defined diagnostic criteria. METHODS: A prospective study was performed on 2,023 consecutive patients between the ages of 6 months and 18 years presenting for an initial comprehensive examination at the Eye Institute of The Pennsylvania College of Optometry. There were 373 subjects between 6 months and 5 years, 11 months of age, and 1,650 subjects between 6 years and 18 years of age. RESULTS: The most important finding from this study is that other than refractive anomalies, the most common conditions optometrists are likely to encounter in a pediatric population are binocular vision and accommodative disorders. The prevalence of accommodative and binocular (strabismic and non-strabismic) vision disorders is 9.7 times greater than the prevalence of ocular disease in children 6 months to 5 years of age, and 8.5 times greater than the prevalence of ocular disease in children 6 to 18 years of age. CONCLUSIONS: The data from this study has great significance for clinicians, optometric educational institutions, health care planners, and administrators. This data suggests that other than refractive anomalies, the most prevalent conditions in the clinical pediatric population are binocular and accommodative disorders. Clinicians should use a minimum data base that includes assessments of accommodation and binocular vision that will allow them to detect conditions with the highest prevalence.

Stereoacuity development: 6 months to 5 years. A new tool for testing and screening.

A new tool to measure changes in random dot stereoacuity development from 6 months to 5 years of age was developed and tested. Either a forced choice or operant preferential looking (PL) paradigm with a happy face target was successfully used to test 136 children. Results indicate that stereoacuity measurements continue to develop through the first years of life. Although children below 24 months of age can be expected to have stereo thresholds in the range of 300 sec arc, there is a transition at approximately 24 months of age after which stereoacuity approaches adult levels. The sensitivity of this test in detecting binocular vision problems was 80%. These results provide guidelines for the assessment of stereoacuity in young children and also provide a new tool for the early detection of binocular vision anomalies.

Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Gö1, both encoding a membrane-bound hydrogenase and a cytochrome b.

DNA encompassing the structural genes of two membrane-bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505-511]. Northern-blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N-terminal amino acid sequences of vhoC and vhtC and the N-terminus of a 27-kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N-terminus of the small subunit indicate the presence of a 48-amino-acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C-terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92-97% identity. Only the C-termini of VhoC and VhtC were not similar.

Optometric management of optically induced consecutive exotropia.

A 5-1/2 year old black female initially presenting with a moderate angle esotropia and latent hyperopia developed a large angle constant exotropia 2 years after final correction of her refractive error. The occurrence of consecutive exotropia as a result of optical correction of hyperopia has been documented infrequently in the ophthalmologic literature and has rarely been mentioned in the optometric literature. While the overall risk for occurrence of this complication from correction of hyperopia may be small, it is a problem which may occur and can be avoided. Unfortunately, there is only limited information about the various risk factors that should be monitored to avoid the occurrence of an optically induced consecutive exotropia. What is available with regard to evaluation and management is scant, and there are no case reports emphasizing optometric management which includes the use of lenses, occlusion and vision therapy. The purpose of this paper is to present a case report of optically induced consecutive exotropia followed by a summary of the available information from the optometric and ophthalmologic literature. This combined information will aid optometrists managing these patients to avoid the occurrence of this problem and better understand the various management aspects when it does occur.

Production of Thermostable alpha-Amylase, Pullulanase, and alpha-Glucosidase in Continuous Culture by a New Clostridium Isolate.

The production of alpha-amylase, pullulanase, and alpha-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60 degrees C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of alpha-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.

Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation.

Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo 32P-labeled C. sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi [32]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation.

Characterization of ATP citrate lyase from Chlorobium limicola.

ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola.