Autonomic and metabolic effects of OSA in childhood obesity.

This study investigates the effects of exposure to intermittent hypoxia on cardiovascular autonomic control and metabolic function in obese children with obstructive sleep apnea (OSA). Each subject underwent: (1) a polysomnography; (2) morning fasting blood samples and a subsequent FSIVGTT; (3) noninvasive measurement of respiration, arterial blood pressure, and heart rate during supine and standing postures. Assessment of adiposity was performed using a DEXA scan. From these measurements, we deduced the pertinent sleep parameters, Bergman minimal model parameters and the parameters characterizing a minimal model of cardiovascular variability. Results suggest that intermittent hypoxia in OSA contributes independently to insulin resistance and autonomic dysfunction in overweight children.

Loss of THCCOOH from urine specimens stored in polypropylene and polyethylene containers at different temperatures.

The loss of delta9-tetrahydrocannabinol (THCCOOH) from urine specimens stored in polypropylene and polyethylene containers at 4 degrees C and 25 degrees C was examined. All specimens were analyzed by GC-MS after sampling at various times over a one-week period. Data were analyzed by one-way analysis of variance and fitted with a first order kinetic equation. Rapid loss of THCCOOH was seen at 4 degrees C for both polypropylene (14% maximal loss, t(1/2) = 0.53 min) and polyethylene (17% maximal loss, t(1/2) = 5.77 min) bottles. At 25 degrees C, a small loss (< 5%) was observed in polypropylene and no significant loss was seen for urine in polyethylene. All losses stabilized within 1 h, and no further losses were seen over one week. The results indicate that THCCOOH binding may be due to decreased solubility of THCCOOH at lower temperatures and subsequent association of THCCOOH with the more lipophilic plastic. The results also indicate that polypropylene and polyethylene do not bind THCCOOH to such an extent as to compromise the integrity of specimens.

Two outbreaks of trichinellosis in the state of Northrhine-Westfalia, Germany, 1998.

Illness or death from trichinellosis is statutorily notifiable in Germany. Between nought and ten cases were reported each year from 1987 to 1997. From November 1998 to January 1999, however, 52 cases of trichinellosis were identified by the public health

Effective GC-MS procedure for detecting iso-LSD in urine after base-catalyzed conversion to LSD.

A sensitive method is described to detect isolysergic acid diethylamide (iso-LSD) in urine. The compound was extracted from urine and converted to a C-8 carbanion by sodium ethoxide in ethanol. Protonation of the carbanion by water selectively produced LSD. The conversion of iso-LSD to LSD was almost quantitative (98%). The product was purified by solid-phase fractionation and acid-base separation techniques. The trimethylsilyl derivative of LSD was detected by a gas chromatography-mass spectrometry method. The overall recovery of the procedure was approximately 69%. Quantification of iso-LSD was linear over the concentration range 50-2000 ng/L. In specimen analysis, iso-LSD was detected when the LSD concentration was below the limit of the detection (50 ng/L) of the procedure. Because iso-LSD is a byproduct of illicit preparation of LSD, presence of iso-LSD in urine is an indication of LSD use.

Structural adaptations in the interaction of EcoRI endonuclease with methylated GAATTC sites.

We have studied the interaction of EcoRI endonuclease with oligonucleotides containing GAATTC sites bearing one or two adenine-N6-methyl groups, which would be in steric conflict with key protein side chains involved in recognition and/or catalysis in the canonical complex. Single-strand methylation of either adenine produces small penalties in binding free energy (deltadeltaG0(S) approximately +1.4 kcal/mol), but elicits asymmetric structural adaptations in the complex, such that cleavage rate constants are strongly inhibited and unequal in the two DNA strands. The dependences of cleavage rate constants on the concentration of the Mg2+ cofactor are unaltered. When either adenine is methylated on both DNA strands, deltadeltaG0(S) (approximately +4 kcal/mol) is larger than the expected sum of the deltadeltaG0(S) values for the single-strand methylations, because the asymmetric adaptations cannot occur. Cleavage rate constants are reduced by 600 000-fold for the biologically relevant GAmATTC/CTTmAAG site, but the GmAATTC/CTTAmAG site forms only a non-specific complex that cannot be cleaved. These observations provide a detailed thermodynamic and kinetic explanation of how single-strand and double-strand methylation protect against endonuclease cleavage in vivo. We propose that non-additive effects on binding and structural 'adaptations' are important in understanding how DNA methylation modulates the biological activities of non-catalytic DNA binding proteins.

Facilitated distortion of the DNA site enhances EcoRI endonuclease-DNA recognition.

We have measured the binding of EcoRI endonuclease to a complete set of purine-base analogue sites, each of which deletes one functional group that forms a hydrogen bond with the endonuclease in the canonical GAATTC complex. For five of six functional group deletions, the observed penalty in binding free energy is +1.3 to +1.7 kcal/mol. For two of these cases (replacement of adenine N7 with carbon) a single protein-base hydrogen bond is removed without deleting an interstrand Watson-Crick hydrogen bond or causing structural "adaptation" in the complex. This observation establishes that the incremental energetic contribution of one protein-base hydrogen bond is about -1.5 kcal/mol. By contrast, deletion of the N6-amino group of the inner adenine in the site improves binding by -1.0 kcal/mol because the penalty for deleting a protein-base hydrogen bond is outweighed by facilitation of the required DNA distortion ("kinking") in the complex. This result provides direct evidence that the energetic cost of distorting a DNA site can make an unfavorable contribution to protein-DNA binding.

Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex.

We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.

Vasopressin and oxytocin in brain areas of rats selectively bred for differences in behavioral performance.

Wistar rats were selectively bred over 10 generations for differences in performance in a footshock-motivated brightness discrimination (BD) test in a Y-maze. High behavioral performance (Wis/HBP) and low behavioral performance (Wis/LBP) rat lines were obtained which differ significantly in all behavioral components tested: frequency of correct responses, number of trials to criterion, response latency (HBP less than LBP), and frequency of freezing behavior (HBP less than LBP), the latter suggesting differences in emotionality. In Wis/LBP rats, furthermore, the normal increase in behavioral performance between the training and the relearning session, which indicates the formation of a memory trace, disappeared during selection. In male breeders sampled during selection of the two lines (Wis/HBP: n = 17; Wis/LBP: n = 21), both arginine vasopressin (AVP) and oxytocin (OXT) contents were measured by radioimmunoassay in the motor cortex, septum/striatum, hippocampus, hypothalamus, medulla oblongata and posterior pituitary. Compared with the Wis/HBP rats, the Wis/LBP rats contained less AVP in the hippocampus (3.1 +/- 0.58 vs 8.3 +/- 1.4 pg/mg wet wt., mean +/- S.E.M., P less than 0.001), but more AVP in the medulla (1.7 +/- 0.20 vs 1.1 +/- 0.18 pg/mg, P less than 0.05). In contrast, no significant differences between the lines were detected with respect to OXT concentrations. In the Wis/LBP rats, moreover, the hippocampal AVP content decreased during selection (r = -0.645, P less than 0.01), while the acquisition response latency increased (r = 0.549, P less than 0.01). As a consequence, a significant, albeit weak, negative correlation (r = -0.483, P less than 0.05) was observed between the individual hippocampal AVP content and the response latency during acquisition. Thus, the results confirm the view that genetically determined differences in the hippocampal content of endogenous AVP may contribute to an individual's level of emotionality and behavioral performance.

The energetic basis of specificity in the Eco RI endonuclease--DNA interaction.

High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence.

"Ultraviolet footprinting" accurately maps sequence-specific contacts and DNA kinking in the EcoRI endonuclease-DNA complex.

The "UV footprinting" technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure of the EcoRI endonuclease-DNA complex allows us to determine how UV light detects protein-DNA contacts. We find that kinking of the DNA helix in the complex greatly enhances the UV photoreactivity of DNA at the site of the kink. In contrast to kinking, contacts between the endonuclease and the DNA bases inhibit the UV photoreactivity of DNA. Similar analysis of a proteolytically modified endonuclease that exhibits the same sequence specificity as wild-type enzyme but that does not cleave DNA supports these conclusions. Furthermore, detection of enhanced photoreactivity at the same kink in the modified enzyme-DNA complex allows us to conclude that the loss of cleavage activity by the modified endonuclease is not due to its failure to kink DNA.

The enfolding arms of EcoRI endonuclease: role in DNA binding and cleavage.

The N-terminal segments of the EcoRI endonuclease dimer form part of mobile "arms" that encircle DNA in the recognition complex. By treating endonuclease-TCGCGAATTCGCG complexes with proteases, we have prepared a series of deletion derivatives lacking defined segments of the N-terminal region. The 5-12 segment is essential for DNA cleavage and forms one electrostatic interaction (per subunit) with DNA phosphate. These ionic contacts are directly across the double helix from the scissile phosphodiester bonds; they thus may permit the enfolding arms to immobilize DNA in apposition to the catalytic cleft and/or contribute to the unusual "kinked" conformation of DNA in the complex. Sequence specificity is fully retained when 28 residues are deleted from the N-terminus, but the complexes dissociate more rapidly.

Two-fold symmetry of crystalline DNA-EcoRI endonuclease recognition complexes.

Recognition complexes between EcoRI endonuclease and either of two synthetic oligonucleotides (sequences CGCGAATTCGCG and TCGCGAATTCGCG) crystallize in Space Group P321 with unit cell parameters a = 128 and c = 47 A and a = 118.4 and c = 49.7 A, respectively. Native diffraction data to 3 A resolution have been collected from the form containing the tridecameric sequence. Electrophoretic analyses of dissolved crystals demonstrate that this form contains DNA and protein in a ratio of one double helix per enzyme dimer. The most likely asymmetric unit contents are one 31,000 dalton enzyme subunit and one strand of DNA, yielding VM values of 3.1 A3/dal and 2.8 A3/dal for the forms containing dodecameric and tridecameric DNA, respectively. This implies that the DNA-protein complex possesses two-fold rotational symmetry, which has been incorporated in the crystalline lattice.

Coordinate ion pair formation between EcoRI endonuclease and DNA.

The free energy of the binding reaction between EcoRI restriction endonuclease and a specific cognate dodecadeoxynucleotide (d(CGCGAATTCGCG)) has contributions from both electrostatic and nonelectrostatic components. These contributions were dissected by measuring the effects of varying salt concentration on the equilibrium binding constant and applying the thermodynamic analyses of Record et al. (Record, M. T., Jr., Lohman, T. M., and deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158). Endonuclease mutation S187 (Arg 187 to Ser) (Greene, P. J., Gupta, M., Boyer, H. W., Brown, W. E., and Rosenberg, J. M. (1981) J. Biol. Chem. 256, 2143-2153) did not significantly affect the nonelectrostatic component but did perturb the electrostatic contribution to the binding energy (we are numbering the amino acid residues according to the DNA sequence). The former was determined by extrapolating the linear portion of the salt dependence curve (0.125 to 0.25 M KCl) to 1 M ionic strength, with the same result for both wild type and S187 endonucleases at both pH 6.0 and 7.4 (-8.5 +/- 1.5 kcal/mol or greater than 50% of the total binding free energy). The slopes of these same curves yield estimates of eight ionic interactions between wild type endonuclease and the DNA at both pH values. By contrast, binding of EcoRI-S187 to dodecanucleotide involves six charge-charge interactions at pH 6.0. Only two ionic interactions are observed at pH 7.4. This was unexpected since gel permeation chromatography demonstrated that the recognition complex for both wild type and S187 proteins contains an enzyme dimer and a DNA duplex. EcoRI-S187 endonuclease retains wild type DNA sequence specificity, and the rate of the phosphodiester hydrolysis step is also unchanged. Thus, electrostatic interactions are functionally separable from sequence recognition and strand cleavage. Our results also establish that arginine 187 plays a key role in the electrostatic function and suggest that it might be located at the DNA-protein interface. The disproportionate loss of ion pairs at pH 7.4 can be rationalized by a model which suggests that six conformationally mobile ionic groups on the protein act in a coordinated manner during the interaction with DNA.

Vasovasostomy: four year experience at Ochsner Medical Institutions.

PIP: Success with the optical loupe magnification system encouraged this report of 21 patients undergoing vasovasostomy from 2-21 years since vasectomy using this procedure. 20 of the 21 patients were available for follow-up. All had undergone single-layer vasovasostomy using optical magnification and fine absorbable suture at the Ochsner Medical Institutions (1975-1979). Ejaculates contained sperm in 18 of the 20 patients. This yielded a patency rate of 90%. Semen analysis revealed a sperm count of greater than 20 million/ml, and average motility rate of 50% with normal movement graded on a scale of 1-4 (normal=2+) in 83% of successful anastomosis. There were 13 pregnancies reported in the postvasovasostomy period, resulting in a pregnancy rate of 68%. 1 patient is single (13 of 19=68%). 2 cases of multiple pregancies were reported. In the group with a 2-5 year vasectomy reversal interval, 8 of 8 patients had patent tubes. In the group 6-10 postvasectomy, 6 of 7 were patent. In the 10 year + group, 4 of 5 had patent tubes.^ieng