Neuroanatomical basis for first- and second-order representations of bodily states

Changes in bodily states, particularly those mediated by the autonomic nervous system, are crucial to ongoing emotional experience. A theoretical model proposes a first-order autoregulatory representation of bodily state at the level of dorsal pons, and a second-order experience-dependent re-mapping of changes in bodily state within structures such as cingulate and medial parietal cortices. We tested these anatomical predictions using positron emission tomography and a human neurological model (pure autonomic failure), in which peripheral autonomic denervation prevents the emergence of autonomic responses. Compared to controls, we observed task-independent differences in activity of dorsal pons and context-induced differences in cingulate and medial parietal activity in PAF patients. An absence of afferent feedback concerning autonomically generated bodily states was associated with subtle impairments of emotional responses in PAF patients. Our findings provide empirical support for a theory proposing a hierarchical representation of bodily states.     

An Extension of the Regression of Offspring on Mid-Parent to Test for Association and Estimate Locus-Specific Heritability: The Revised ROMP Method

The Regression of Offspring on Mid-Parent (ROMP) method is a test of association between a quantitative trait and a candidate locus. ROMP estimates the trait heritability and the heritability attributable to a locus and requires genotyping the offspring only. In this study, the theory underlying ROMP was revised (ROMP_rev) and extended. Computer simulations were used to determine the type I error and power of the test of association, and the accuracy of the locus-specific heritability estimate. The ROMP_rev test had good power at the 5% significance level with properly controlled type I error. Locus-specific heritability estimates were, on average, close to simulated values. For non-zero locus-specific heritability, the proposed standard error was downwardly biased, yielding reduced coverage of 95% confidence intervals. A bootstrap approach with proper coverage is suggested as a second step for loci of interest.
ROMP_rev was applied to a study of cardiovascular-related traits to illustrate its use. An association between polymorphisms within the fibrinogen gene cluster and plasma fibrinogen was detected (p < 0.005) that accounted for 29% of the estimated fibrinogen heritability. The ROMP_rev method provides a computationally fast and simple way of testing for association and obtaining accurate estimates of locus-specific heritability while minimizing the genotyping required.     

Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis

The definition of the temporal sequence of appearance of fetal markers during prenatal and early postnatal development in Sertoli and germ cells may be important for understanding the mechanisms underlying their reexpression in disorders of the adult testis. For this reason, we studied the expression of Sertoli and germ cell markers in 25 human testes spanning a period from 8 gestational weeks to 4 years. Well-characterized antibodies were employed to anti-Müllerian hormone (AMH), cytokeratin 18 (CK18), vimentin (VIM), M2A-antigen (M2A), germ cell alkaline phosphatase (GCAP), and somatic angiotensin-converting enzyme (sACE) on formalin-fixed and microwave-pretreated paraffin sections. In Sertoli cells, AMH and VIM were consistently present. While VIM and CK18 were coexpressed in embryonic testes, CK18 was progressively downregulated and completely absent from the 20th gestational week. M2A was absent or moderately expressed in fetal Sertoli cells but increased during further development. In germ cells, M2A was consistently found in primordial germ cells (PGCs) as well as in M- and T₁-prespermatogonia. In contrast, sACE and GCAP were absent from PGCs but were a distinct feature of late M- and early T₁-prespermatogonia and appeared predominantly between the 18th and the 22nd gestational weeks. Both T₂-prespermatogonia and postnatal prespermatogonia were devoid of any marker. While CK18 represents a differentiation marker for fetal Sertoli cells, M2A, GCAP, and sACE can be used as differentiation markers for the discrimination of different germ cell types during human prespermatogenesis. Because various immunophenotypes reflect distinct differentiation stages, this knowledge may be important for understanding adult testicular pathology.     

Efficacy of pyrimethamine/sulfadoxine in the prevention of toxoplasmic encephalitis relapses andPneumocystis carinii pneumonia in HIV-infected patients

The efficacy and safety of 25 mg pyrimethamine plus 500 mg sulfadoxine given twice a week in preventing relapses of AIDS-related toxoplasmic encephalitis was evaluated in an open study. The 56 HIV-infected patients evaluated had responded to intensive treatment with pyrimethamine/clindamycin prior to starting the present prophylactic regimen. Four patients (7 %) experienced relapse while on pyrimethamine/sulfadoxine. The probability of freedom from relapse was >90 % for 12 months and >80 % for 24 months. Side effects comprised mild or moderate allergic reactions which occurred in 23 patients (41 %), leading to discontinuation in four patients (7%). Forty-nine of the 56 patients did not have a history ofPneumocystis carinii pneumonia and did not receive antiparasitic prophylaxis other than pyrimethamine/sulfadoxine; two of them (4 %) developed pneumocystosis. The probability of freedom from pneumocystosis was about 90 % for 24 months. Pyrimethamine/sulfadoxine twice a week appears to be a promising regimen for prevention of toxoplasmic encephalitis, and also appears to provide protection againstPneumocystis carinii pneumonia. Although allergic reactions are usually mild and disappear on continuation, they may limit the value of this regimen.     

Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA

During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Since patients undergoing testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) reveal low fertilization rates, whether the outcome of ICSI could be related to the percentage of round spermatids expressing PRM1-mRNA and PRM2-mRNA was investigated. Applying in-situ hybridization, 55 testicular biopsies from men undergoing TESE/ICSI were investigated. The percentage of PRM1-mRNA and PRM2-mRNA positive spermatids was significantly (P < 0.0001) decreased in men with at least qualitatively normal spermatogenesis (PRM1-mRNA: 58.4 +/- 13.8%; PRM2-mRNA: 56.4 +/- 11.3%) and impaired spermatogenesis (PRM1-mRNA: 32.6 +/- 10.8%; PRM2-mRNA: 31.7 +/- 11.1%) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (PRM1-mRNA: 79.9 +/- 4.6%; PRM2-mRNA: 78.1 +/- 5.7%). A positive correlation (r(PRM1) = 0.733; r(PRM2) = 0.784; P < 0.001) was demonstrated between the score and the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids. While successful fertilization was neither related to the score, nor to the percentage of PRM1-mRNA and PRM2-mRNA positive spermatids, a significant (P < 0.05) relationship was demonstrated between successful fertilization and the PRM1-mRNA to PRM2-mRNA ratio. Therefore, the PRM1-mRNA to PRM2-mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI.     

Biological and clinical significance of cytogenetic abnormalities in low-risk and high-risk gastrointestinal stromal tumors

We report cytogenetic findings in 19 c-Kit-positive gastrointestinal stromal tumors (GISTs) that represent a heterogenous group of mesenchymal neoplasms with respect to site, histology, and biologic behavior. All of the GISTs (5 low-risk, 11 high-risk, 3 recurrences) displayed clonal chromosomal aberrations; 15 were hypo- to near-diploid, and 4 were near-triploid and hypotetraploid. The most common abnormalities were loss of chromosomes 14 and/or 22, demonstrated in 14 GISTs irrespective of site or predominant phenotype. Ten cases (2 low-risk, 5 high-risk, 3 recurrences) were characterized by loss of both chromosomes 14 and 22, 2 cases (1 low-risk, 1 high- risk), by loss of chromosome 14; and 2 high-risk cases, by loss of chromosome 22. Additional chromosomal aberrations occurred preferentially in high-risk and recurrent GISTs, including loss of 9p and 1p in 8 cases each, loss of 15 in 6 cases, loss of 3p in 5 cases, loss of 13q and 10q in 4 cases each, loss of 19 in 3 cases, and complete or partial gains of chromosomes 5 and 4 in 2 cases each. More significantly, 5 of 6 patients with clinically aggressive GISTs, including 2 recurrences and 3 metastasing GISTs, were additionally characterized by loss of 9p; four of these had additional loss of chromosomes 1p and 15. The presented results herein indicate that loss of chromosome 14 and/or 22 is an early change in GIST tumorigenesis irrespective of site or differentiation, whereas malignant transformation and progression of GISTs appear to be associated with an increasing incidence of additional secondary aberrations.     

Facial Muscle Tonus During REM and NREM Sleep

Equipment and procedures were devised for quantifying relatively noise-free recordings of low amplitude chin and lip electromyograms (EMGs) during sleep. A total of 28 REM periods were recorded from 5 young adult female Ss. Tonic EMG levels tended to decline toward their lowest level of the night beginning 5 min in advance of REM periods. With very rare exceptions, the lowest EMG levels of the night were maintained throughout REM sleep. During the 20 min of NREM sleep which followed REM periods, mean EMG levels increased over the REM levels but were lower than those recorded during the 20 min of NREM sleep which preceded the REM periods. This pattern of tonic EMG variation obtained for each of the first 3 REM periods of the night.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.