Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.     

MRI of the spine in cobalamin deficiency: the value of examining both spinal cord and bone marrow

Low back pain is caused by a variety of etiologies. Some clinicians have postulated that much low back pain is due to trauma to the iliolumbar ligament. The iliolumbar ligament is one of the three pelvic-lumbar ligaments and develops during the 12th week of gestation. The iliolumbar ligament appears to be a major stabilizing component between the vertebral spine and the pelvis. The innervation of the iliolumbar ligament appears similar to the posterior lumbar ligaments. Our hypothesis is: micro-trauma to the iliolumbar ligament is the primary cause of many cases of chronic low back pain because (1) it is the weakest component of the multifidus triangle; (2) there is increased susceptibility to injury due to its angulated attachment; (3) it is a primary inhibitor of excess sacral flexion; (4) it is a highly innervated nociceptive tissue; and (5) it plays an increased role with progressive disc degeneration.     

Importance of participation rate in sampling of data in population based studies, with special reference to bone mass in Sweden

OBJECTIVE: To study the effects of participation rate in sampling on "normative" bone mass data. DESIGN: This was a comparison between two randomly selected samples from the same population. The participation rates in the two samples were 61.9% and 83.6%. Measurements were made of bone mass at different skeletal sites and of muscle strength, as well as an assessment of physical activity. SETTING: Malm?, Sweden. SUBJECTS: There were 230 subjects (117 men, 113 women), aged 21 to 42 years. RESULTS: Many subjects participated in both studies (163). Those who took part only in the study with the higher participation rate (67) almost invariably had higher values for bone mass density at the sites measured (up to 7.6% for men) than participants in the study with the lower participation rate. No differences in muscle strength were recorded. CONCLUSION: A high degree of compliance is important to achieve a reliable result in determining normal values in population based studies.     

Clonal analysis of bilateral breast cancer

Forty-nine pairs of bilateral breast tumors (41 synchronous and 8 asynchronous cases) were examined for X-chromosome inactivation status and p53 mutations to address the issue of their clonality. Among 12 cases that were informative for the trinucleotide repeat polymorphism in exon 1 of the androgen receptor gene on the X chromosome, 3 cases were found to have different alleles of the locus inactivated in the right and left breast tumors, indicating that the two tumors arose from distinct transformed cells. Thirteen tumors (13%) from 11 women (22%) contained somatic mutations in exons 5-8 of the p53 gene. In two cases, both breast tumors harbored p53 mutations, but the specific mutations were not identical. Seven synchronous and two asynchronous cases had p53 mutations in one tumor only. A germ line p53 mutation at codon 248, one of the most common p53 mutations in Li-Fraumeni syndrome, was observed in one case. Immunohistochemical analysis of p53 protein with a monoclonal antihuman p53 antibody showed concordant positivity between the right and left tumors in three bilateral breast cancer cases. Our results suggest that at least some bilateral breast tumors originate from distinct cells, but that some bilateral breast tumors may be related through a common p53 abnormality.     

The effects of inclusion on the social functioning of students with learning disabilities

The purpose of this study was to provide data on the social functioning (i.e., the degree of peer acceptance, self-concept, loneliness, and social alienation) of students in second, third, and fourth grade who participated in an inclusive classroom for an entire year. The social functioning of students identified as learning disabled (LD; n = 16), low achieving (LA; n = 27), and average/high achieving (AHA; n = 21) was assessed at the beginning and end of the school year. The students with LD were less well liked and more frequently rejected than AHA students. Although students' overall self-worth did not differ by achievement group, the students with LD demonstrated significantly lower academic self-concept scores. The students with LD did not differ on ratings of loneliness, and they demonstrated increases in the number of within-class reciprocal friendships from fall to spring. Discussion focuses on the effects of inclusion on the social functioning of students with LD.     

Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by -20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and 3H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation.     

trans-2-Aryl-N,N-dipropylcyclopropylamines: synthesis and interactions with 5-HT(1A) receptors

Twelve N,N-dipropyl-substituted derivatives of trans-2-arylcyclopropylamine have been prepared and assayed for their ability to displace [(3)H]-8-OH-DPAT from rat brain 5-HT(1A) receptors. The new derivatives include phenyl (7a), bromo- (7b) and fluorophenyl (7c-e), 2-methoxy-5-fluorophenyl (7h), and 2-hydroxy-5-fluorophenyl (7l) as well as trifluoromethylphenyl (7f) and 2,3-dichlorophenyl (7g) analogues. In the present series of compounds, electron-withdrawing substituents in the phenyl ring appear to decrease the affinity for 5-HT(1A) receptors. In contrast, electron-rich aryl groups, such as 2- or 3-thienyl (7j and 7k, respectively), provide compounds with high affinity. The additional bulk produced by the aromatic moiety in the 2-benzothienyl derivative 7i appears to be detrimental to 5-HT(1A) receptor affinity. The racemic mixtures of the interesting 7j and 7l were resolved into the enantiomers; 7j and 7l exhibited a high enantiomeric 5-HT(1A) receptor affinity ratio (75-fold and 100-fold, respectively). The enantiomers of 7j and 7l were evaluated in vivo by use of biochemical and behavioral tests in rats. Compound (1R,2R)-7j behaved as a partial agonist whereas (1R,2S)-7l appeared as an efficacious 5-HT(1A) receptor agonist, stimulating both autoreceptors and postsynaptic receptors.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Bad ethics, good ethics and the genetic engineering of animals in agriculture

PURPOSE: The purpose of this retrospective study is to compare the measurements of intrapapillary and peripapillary parameters between two observers and test the usefulness of measuring different types of crescents. METHODS: Optic disc photographs of 23 eyes of 23 patients with glaucoma and 23 age-matched normal eyes were measured in Oulu and in Erlangen using manual planimetric techniques. The authors measured the following magnification corrected intrapapillary and peripapillary areas: optic disc, neuroretinal rim, cup: disc area ratio, scleral ring, central (zone beta), and peripheral peripapillary atrophy (zone alpha). Twenty-one patients with glaucoma had a follow-up of 3.2 years (range, 1.1-4.7 years), and follow-up for 19 control eyes was 3.7 years (range, 2.5-5.9 years). The measurements were performed in a masked fashion for the diagnosis and temporal sequence of the photographs. RESULTS: Central peripapillary atrophy (zone beta) was statistically significantly largest in primary open-angle glaucoma in both centers (Oulu, P=0.003; Erlangen, P=0.004), whereas normal and exfoliative eyes did not differ significantly from each other. The results for peripheral peripapillary atrophy (zone alpha) and scleral ring were less consistent. Despite statistically significant interobserver correlations ranging from r=0.30 (scleral ring area; P=0.0472) to r=0.97 (optic disc area; P=0.0001), the means of all parameters, except for zone alpha and beta, differed statistically significantly between the two observers. CONCLUSIONS: The central peripapillary atrophy, or zone beta, is the most reproducible parameter when measuring peripapillary atrophy in glaucoma. Nonetheless, its measurement is of limited usefulness in the recognition of glaucoma or progression of glaucomatous nerve damage.