Chromosome territories have a highly nonspherical morphology and nonrandom positioning.

Interphase chromosomes are organized into discrete chromosome territories (CTs) that may occupy preferred sub-nuclear positions. While chromosome size and gene density appear to influence positioning, the biophysical mechanisms behind CT localization, especially the relationship between morphology and positioning, remain obscure. One reason for this has been the difficulty in imaging, segmenting, and analyzing structures with variable or imprecise boundaries. This prompted us to develop a novel approach, based on the two-dimensional (2D) wavelet-transform modulus maxima (WTMM) method, adapted to perform objective and rigorous CT segmentation from nuclear background. The wavelet transform acts as a mathematical microscope to characterize spatial image information over a continuous range of size scales. This multiresolution nature, combined with full objectivity of the formalism, makes it more accurate than intensity-based segmentation algorithms and more appropriate than manual intervention. Using the WTMM method in combination with numerical simulation models, we show that CTs have a highly nonspherical 3D morphology, that CT positioning is nonrandom, and favors heterologous CT groupings. We discuss potential relationships between morphology, positioning, chromosomal function, and instability.

Antibiotic susceptibility of attached and free-floating Helicobacter pylori.

Helicobacter pylori are found attached to mucous cells of the human stomach or under the mucous layer. Models mimicking the in vivo situation may be more suitable for H. pylori MIC determinations than traditional agar dilution methods. Megraud et al. (Antimicrobial Agents and Chemotherapy 1991, 35, 869-72) developed a model for measuring the susceptibility of attached and free-floating H. pylori. We have modified this model so that free-floating and attached H. pylori are treated in a more similar manner, before and after incubation with antibiotic, and performed additional controls to ensure H. pylori and tissue culture cells are not detrimentally affected and maintain their viability during the course of the experiment. We found only 10% of plate-grown H. pylori were competent for attachment to HEp-2 cells; however, all progeny of attached bacteria remained adherent. Killing curves were performed using 0, 0.001, 0.01, 0.1 and 1 mg/L amoxycillin, and 0, 0.0025, 0.0075 and 0.01 mg/L clarithromycin. H. pylori divided at concentrations

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli.

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

Modulation of the substrate specificity of Escherichia coli dimethylsulfoxide reductase.

X-ray crystallographic studies of the Rhodobacter sphaeroides dimethyl sulfoxide (Me2SO) reductase [Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K. V., & Rees, D. C. (1996) Science, 272, 1615-1620] indicated that the active site is at the bottom of a 25-A funnel. Substrates must travel to the bottom of the funnel for reduction to occur. The homologous DmsA subunit of the trimeric Escherichia coli Me2SO reductase, was subjected to site-directed mutagenesis of residues potentially lining the bottom of the funnel, based on sequence alignment of the E. coli and Rhodobacter Me2SO reductases. Sixteen E. coli DmsA mutants were characterized. Mutants G167N, A178Q, Q179I and R217Q showed functional impairment, as indicated by abnormal anaerobic growth with Me2SO as the sole terminal acceptor, in a recombinant strain deleted for chromosomal dmsABC. The kinetic parameters of the mutant enzymes were examined using the artificial electron donor benzyl viologen and the quinone analogue dimethylnaphthoquinone, with Me2SO and pyridine N-oxide as electron acceptors. Mutants A178Q and R217Q showed dramatic alterations of their electron-acceptor Km, with values at least 35-fold less or greater than wild-type values, respectively, for Me2SO and pyridine N-oxide. T148S showed altered kinetic parameters for pyridine N-oxide and Me2SO, with Km and k(cat) decreasing and increasing approximately fourfold, respectively. Other mutants showed less drastic alterations in kinetic parameters. This analysis has identified amino acids important in substrate binding and catalysis.

Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase.

We have characterized the substrate specificity of dimethyl sulfoxide reductase (DmsABC) of Escherichia coli by determining Km and Kcat values for 22 different substrates. The enzyme has a very broad substrate specificity. The Km values varied 470-fold, while Kcat values varied only 20-fold, implicating Km as the major determinant of Kcat/Km values. Sulfoxides and pyridine N-oxide exhibited the lowest Km values, followed by aliphatic N-oxides. The Kcat values for these compounds also followed the same pattern. Substitution at the 2 or 3 position of the pyridine N-oxide ring had little effect on Km while substitution at the 4 position had a greater effect, and increased Km. Negatively charged substrates were poorly accepted. A few compounds that are not S- or N-oxides were also reduced by the enzyme. Most compounds reduced by DmsABC were not toxic to E. coli under anaerobic growth conditions, and E. coli was able to use many of these compounds anaerobically as terminal electron acceptors in the presence of glycerol. Anaerobic growth on sulfoxides is solely due to DmsABC expression. However, there appears to be another as yet unidentified terminal reductase capable of using pyridine N-oxides as terminal electron acceptors.

Effects of Resistance in White Clover (Trifolium repens) on Heterodera trifolii.

Clones of two partially resistant and two susceptible white clover, Trifolium repens, genotypes were exposed to eggs of Heterodera trifolii and nematode development in stained roots measured at 2, 4, 7, 11, 18, 23, and 37 days after inoculation. The differences in development between nematode populations in resistant and susceptible genotypes showed that resistance operated after infection during feeding and development. At 7 days after inoculation, counts of second-stage juveniles did not differ between genotypes, whereas at 37 days more adults had developed in the susceptible than in the resistant genotypes. In a separate experiment, cysts hosted by susceptible genotypes were larger and contained more eggs than those on resistant genotypes so that the product of the values for cysts per plant and for eggs per cyst resulted in a more sensitive measure of resistance than from using cysts per plant alone.

Association of molybdopterin guanine dinucleotide with Escherichia coli dimethyl sulfoxide reductase: effect of tungstate and a mob mutation.

We have identified the organic component of the molybdenum cofactor in Escherichia coli dimethyl sulfoxide reductase (DmsABC) to be molybdopterin (MPT) guanine dinucleotide (MGD) and have studied the effects of tungstate and a mob mutation on cofactor (Mo-MGD) insertion. Tungstate severely inhibits anaerobic growth of E. coli on a glycerol-dimethyl sulfoxide minimal medium, and this inhibition is partially overcome by overexpression of DmsABC. Isolation and characterization of an oxidized derivative of MGD (form A) from DmsABC overexpressed in cells grown in the presence of molybdate or tungstate indicate that tungstate inhibits insertion of Mo-MGD. No electron paramagnetic resonance evidence for the assembly of tungsten into DmsABC was found between Eh = -450 mV and Eh = +200 mV. The E. coli mob locus is responsible for the addition of a guanine nucleotide to molybdo-MPT (Mo-MPT) to form Mo-MGD. DmsABC does not bind Mo-MPT or Mo-MGD in a mob mutant, indicating that nucleotide addition must precede cofactor insertion. No electron paramagnetic resonance evidence for the assembly of molybdenum into DmsABC in a mob mutant was found between Eh = -450 mV and Eh = +200 mV. These data support a model for Mo-MGD biosynthesis and assembly into DmsABC in which both metal chelation and nucleotide addition to MPT precede cofactor insertion.

The developing cerebral surface. Preliminary report on the patterns of sulcal and gyral maturation--anatomy, ultrasound, and magnetic resonance imaging.

This literature review provides data on the temporal sequence in which gyri, sulci, and fissures first become apparent on anatomic specimens of the developing brain surface. Ultrasonic display of these anatomic features lags behind the initial appearance of the features by a variable interval, greater in early gestation and diminishing in late gestation. MR imaging, especially three-dimensional surface rendering, displays the degree of surface maturation to advantage. Different publications have provided variable estimates of the time course of surface maturation, leading to discordance in the date tabulated. None-the-less, the overall sequence is clear and the surface features are useful in assessing the degree of fetal-neonatal maturity by ultrasonography and by MR imaging.

A pinhole method for measuring chest wall compliance.

We describe a method of measuring chest wall compliance (Cw) that readily detects whether respiratory muscles are relaxed. The method simulates a normal slow sigh, with the subject exhaling through a needle valve. Cw is calculated from the slope of the volume-esophageal pressure line. With relaxed subjects, repeated measurements yield similar slopes. When subjects cannot relax, the volume-pressure line is irregular and variable. In 26 subjects who could relax, Cw averaged 0.208 +/- 0.05 (SD) l/cmH2O.

The epizootiology of nematode parasites of sheep in a high-rainfall area of Zimbabwe.

Fifty-two untreated lambs from a contaminated flock were slaughtered at regular intervals throughout the year, and their gastro-intestinal tracts were examined for the presence of nematode parasites. Haemonchus contortus and Oesophagostomum columbianum were found to be of major importance. The incidence of Haemonchus rose to a peak and remained at a high level throughout the winter through the fourth larval stages of the parasite predominated at this stage. Oesophagostomum columbianum was recovered in numbers unusually high for this species, the incidence of which remained at a relatively high level from March until October. The minor genera recovered were Trichostrongylus spp., Cooperia spp., Strongyloides papillosus and Trichuris ovis.

Microsurgical relationships of the anterior inferior cerebellar artery and the facial-vestibulocochlear nerve complex.

The relationship of the anterior inferior cerebellar artery (AICA) to the facial (7th) and vestibulocochlear (8th) nerves was studied using 3x to 20x magnification in 50 cerebellopontine angles (CPAs) from 25 adult cadavers. The AICA originated from the basilar artery as a single (72% of the CPAs), duplicate (26%), or triplicate (2%) artery. Each of the 50 CPAs had one or more arterial trunks that coursed in close proximity to the 7th and 8th cranial nerves and thus were said to be nerve-related. The nerve-related arterial trunks were divided into three segments based on their relationship to the nerves and meatus: the premeatal, meatal, and postmeatal segments. The meatal segment projected to the meatus or into the canal in 64% of the CPAs. In relation to the nerves, the premeatal segment was most commonly anteroinferior, the meatal segment was inferior, and the postmeatal segment was posteroinferior. The nerve-related branches of the AICA gave rise to internal auditory arteries in 100% of the 50 CPAs, recurrent perforating arteries in 82%, and the subarcuate artery in 72%. The internal auditory and recurrent perforating arteries arose most commonly from the premeatal segment, and the subarcuate artery arose most commonly from the postmeatal segment. There were one to four internal auditory arteries per CPA, zero to three recurrent perforating arteries, and zero or one subarcuate artery. The effects of occlusion of the nerve-related arteries and their involvement in conditions treated by neurosurgeons are reviewed.

Bronchial adenoma resection with relief of hypoxic pulmonary vasoconstriction.

Experimental airway obstruction is known to cause reflex pulmonary artery constriction, but clinical documentation of reversible bronchial obstruction and vasoconstriction is rare. A soft bronchial adenoma obstructed the left main bronchus, and scans showed minimal ventilation and perfusion on the left. Gas aspirated from beyond the tumor was hypoxic. The adenoma was removed and the lung left intact by means of a skin graft in the bronchial wall. Four months later, pulmonary function was normal, and both ventilation and perfusion of the left lung were normal. Reflex pulmonary vasoconstriction resulting from alveolar hypoxia minimizes systemic hypoxemia and also minimizes alveolar tissue hypoxia in the lung itself. The reflex is seen most frequently in perfusion scans in patients with chronic airways disease. This case in important in that it documents reversal of vasoconstriction after ventilation was restored.

Use of biofeedback in weaning paralyzed patients from respirators.

Two paralyzed patients who were being treated with mechanical respirators and in whom weaning with intermittent mandatory ventilation failed were treated with regular sessions of biofeedback. Their respiratory volumes were displayed to them on oscilloscopes at bedside. A plastic (Plexiglas) template was placed on the face of the oscilloscope with two horizontal strips of adhesive tape affixed to it; the patient had to achieve a certain tidal volume (TV) to push the tracing outside the space between the horizontal lines. During each session the highest TV achieved determined the target TV to be maintained during the next session. The application of treatment coincided with improved ventilation; both patients were eventually weaned.

Chronic binodal block with Wenckebach phenomenon.

Coexistent sino-atrial and atrioventricular block with Wenckebach phenomenon is an extremely unusual cardiac arrhythmia. Observations on the natural history of sinus node dysfunction are rare. For over six years our elderly patient has been followed without symptoms but with increasing severity of atrio-ventricular block.