Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii

Summary Genetic analysis of 25 nuclear mutants defective in the chlorophyll-protein complex CP1 was undertaken. The mutants belong to 13 complementation groups scattered throughout the nuclear genome. All these mutants lack the apoprotein of CP1 and, in addition, a specific set of six low molecular weight thylakoid polypeptides. System I particles obtained by treating WT thylakoid membranes with detergent specifically contain those polypeptides which the mutants lack. These observations suggest that a particular sub-structure of the thylakoid membrane associated with the photosystem I activity is missing from all 25 mutants studied, and that this general phenotype can result from mutation at any one of several unlinked Mendelian loci.     

Analysis of very long-chain fatty acids using electrospray ionization mass spectrometry

Elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues are the biochemical hallmark for patients with X-linked adrenoleukodystrophy (X-ALD). Current methods for the determination of VLCFA levels are laborious and time-consuming. We describe a rapid and easy method using electrospray ionization mass spectrometry (ESI-MS) with deuterated internal standards. VLCFA are hydrolyzed, extracted, and quantified in less than 4h. This includes 2h of hydrolysis and 4min of quantification. We validated the method by analyzing 60 plasma samples from controls and patients with X-ALD or Zellweger syndrome using both the ESI-MS protocol and an established method for VLCFA analysis using gas chromatography (GC). The C26:0 concentrations determined with ESI-MS in plasma and fibroblasts of X-ALD patients are in good agreement with those reported previously for GC and GC-MS. Besides saturated straight chain VLCFA, we also determined the concentrations of the mono-unsaturated VLCFA C24:1 and C26:1 and established that while C24:1 levels are not elevated, C26:1 levels are elevated in both plasma and fibroblasts from X-ALD patients.     

Cognitive and motor skills in achondroplastic infants: Neurologic and respiratory correlates

Thirteen infants with achondroplasia underwent psychometric testing as part of a comprehensive neurologic assessment. As a group, mental development was average and motor development was delayed, although a wide range of scores was obtained. Foramen magnum measurements were correlated with respiratory dysfunction, abnormal so-matosensory evoked potentials, and delayed motor development. Abnormal polysomno-gram outcome was associated with reduced mental capacity. In light of the reported increased frequency of respiratory dysfunction in achondroplasia, these findings warrant careful attention and further study.     

T-cell receptor variable region of the β-chain gene use in peripheral blood and multiple synovial membranes during rheumatoid arthritis

In order to look for a site-specific T-cell response in RA SM, PCR analyses using oligonucleotide primers specific for 24 TCRBV (Vβ) families were performed to compare the respective usage of each TCRBV gene by T cells present in PB and SM of 13 patients with RA. In four patients, SM cells from two or three sites of inflammation were subjected to analysis. In one patient, synovial tissue was studied at two different phases of the disease, resulting in a total number of 19 samples of SM cells, which were compared with paired samples of PB cells. The results showed that whereas all 24 TCRBV gene families could be detected in both PB and SM cells, there was some skewing of increased or decreased usage frequencies of particular TCR Vβ genes among SM cells. Three TCRBV families were often overexpressed in SM: Vβ3, Vβl7, and Vβ22. Moreover, Vβ4 was often decreased in SM (7 out of 13). This decrease was statistically significant in the RA population studied. SM from different joints of a given patient showed similar variations of T-cell repertoire compared to PB, even 6 months later in the course of the disease. These results demonstrate a biased TCRBV gene utilization in RA SM. This bias appears to be similar in different joints and at different times in the course of the disease. No correlation was found between the bias of TCR repertoire in SM and the HLA typing of these patients.     

Adolescents'' opinions about genetic risk information, prenatal diagnosis, and pregnancy termination

Advances in genetics create increasing possibilities of diagnosing and preventing genetic disease. In most countries, the community is poorly informed about the role of genetic factors in human disease and about genetic testing and its social, emotional, and ethical implications. School education about genetics may improve this situation. Students are, of course, the adults of the future and the potential users of the new genetic tests. To gain further insight into the perception of genetic risk of adolescents and their perception of the new genetic techniques and as a starting point for setting up an adequate information campaign in Flanders, we assessed the opinions and beliefs of students with regard to health, genetic diseases, genetic risk, and genetic testing.

A standardised interview and questionnaire were administered within the scope of the two yearly medical check up of 166 fifth grade students. They were randomly selected from the group of all fifth grade high school students in seven different schools.

This paper focuses on the attitudes of adolescents towards obtaining genetic information, towards prenatal diagnosis and pregnancy termination. Adolescents in Flanders are interested in being informed about genetic risks and genetic diseases and in making use of prenatal diagnosis because they want to make informed reproductive decisions in the future and to be emotionally prepared for the birth of an affected child. They adopt a critical attitude towards pregnancy termination. The association between these attitudes and several relevant factors was investigated. This showed significant correlations between some attitudes and general health related prevention, perceived burden of genetic diseases, the importance of the value “own health”, the perceived role of society, and the regularity of religious practice. Some points for special attention were formulated with regard to information campaigns for adolescents.

     

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10⁻⁶M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.     

Detailed clinical description of four patients with 1.3 and 2.1 Mb chromosome imbalances derived from a familial t(12;17)(q24.33;q25.3)

We describe a family carrying a submicroscopic reciprocal translocation involving 12qter and 17qter detected by subtelomeric FISH analysis. Four family members inherited unbalanced variants-two cases inherited the derivative chromosome 12 and the other two the derivative chromosome 17. The two individuals with the derivative chromosome 17 showed a distinct phenotype with mild mental retardation in combination with multiple minor malformations, while the phenotype in the cases with the derivative chromosome 12 was milder and only partly concordant. Detailed FISH analysis using 19 BAC clones covering the distal part of chromosome 12q and 17q estimated the imbalances to 2.1 and 1.3 Mb, respectively. The clinical and cytogenetic findings of the two different genotypes are reported and discussed. This family illustrates that small chromosome imbalances can be detected in individuals with mild phenotype and normal, or near-normal, cognitive functions.     

Ionic basis of membrane potentials of epithelial cells in rat small intestine

1. Potentials across the mucosal and serosal membranes of the epithelial cells of rat jejunum together with transmural potentials were recorded using everted sac preparations.2. Ionic changes in either mucosal or serosal fluids affect mucosal or serosal membrane potentials respectively with comparable changes in the transmural potential. The contralateral membrane potential is relatively unaffected.3. Replacement of mucosal sodium chloride by potassium chloride or lithium chloride had little effect on potentials, but its replacement by mannitol or Tris chloride increased the negativity of the mucosal potential, giving linear relationships against log(10)[Na](m) with slopes of 41.4 and 30.7 mV respectively for tenfold change in [Na](m).4. At constant [Na](m), potassium or lithium increased the mucosal potential by 25.7 and 19.8 mV respectively for tenfold concentration changes.5. Qualitatively similar changes occurred in the serosal potential when the ionic composition of the serosal fluid was varied.6. Mucosal potential changes in response to modifications of the ionic composition of the mucosal fluid were the same in the presence and absence of galactose.7. Sodium and potassium diffusion potentials largely determine both the mucosal and serosal membrane potentials. For the mucosal membrane, P(K):P(Na) is 1.26:1, and is probably higher for the serosal membrane. Chloride makes no significant contribution to membrane potentials.8. Potentials generated by the electrogenic sodium pump are superimposed on diffusion potentials across the serosal membrane.