Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man

Summary Two 46,XY females with tandem duplications of an X short arm segment were studied by cytogenetic and Southern blot analysis. The results show that the duplicated segment in each case included the Xp21.2–Xp22.2 interval, resulting in a double dose of ZFX on the single active X chromosome. The results from our two cases, in conjunction with those reported by other workers, lead us to conclude that the duplication is the reason for the sex inversion. If ZFY and ZFX are indeed sex-determining gene loci, these findings favour a model of sex determination characterized by antagonistic interaction between these genes.     

Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201.

Whole-cell protein patterns of a psychrotrophic Bacillus cereus strain from cultures grown at 7 and 30 degrees C were compared. This analysis revealed that at least three major proteins are expressed at a significantly higher rate at 7 degrees C than at 30 degrees C. The most abundant of these cold-induced proteins was a small polypeptide of 7.5 kDa, designated CspA, of B. cereus. In addition, four small proteins very similar in size to CspA were seen on both 7 degrees C and 30 degrees C two-dimensional protein gels. Immunoblot analysis using B. cereus anti-CspA antibodies indicated that the five proteins described above plus an additional sixth protein not visible on silver-stained two-dimensional gels are members of a B. cereus cold shock protein family. This hypothesis was corroborated by cloning and sequencing of the genes encoding five proteins of this family. The protein sequences deduced are highly similar and show homology to small procaryotic cold shock proteins and to the cold shock domain of eucaryotic Y-box proteins. Besides CspA, only one of the additional five CspA homologs was slightly cold inducible. In the presence of 100 mM NaCl, the two purified members of the protein family (CspA and CspE) elute as dimers at an apparent molecular mass of 15 kDa from a gel filtration column. At higher salt concentrations, they dissociate into their monomers. Their ability to bind to the ATTGG motif of single-stranded oligonucleotides was demonstrated by band shift analysis.     

Characterization of inositolpolyphosphate binding to myocardial membranes

Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP₃) and inositol-1,3,4,5-P₄ (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP₆). This study demonstrates that InSP₆ has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively.InsP₄ binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP₃, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP₄ to SR and SL by a mean of 30% and 20% respectively.Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP₆ bound to two separate protein peaks in both these membranes, while InsP₄ bound to only one. In SR membranes, InsP₄ bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.     

Nucleotide sequence and taxonomical distribution of the bacteriocin gene lin cloned from Brevibacterium linens M18.

Linocin M18 is an antilisterial bacteriocin produced by the red smear cheese bacterium Brevibacterium linens M18. Oligonucleotide probes based on the N-terminal amino acid sequence were used to locate its single copy gene, lin, on the chromosomal DNA. The amino acid composition, N-terminal sequence, and molecular mass derived from the nucleotide sequence of an open reading frame of 798 nucleotides coding for 266 amino acids found on a 3-kb BamHI restriction fragment correspond closely to those obtained from the purified protein (N. Valdés-Stauber and S. Scherer, Appl. Environ. Microbiol. 60:3809-3814, 1994). No sequence homology to any protein or nucleotide sequences deposited in databases was found. Comparison of the nucleotide sequence and the N-terminal amino acid sequence derived from the protein suggests that B. linens M18 produces an N-formyl-methionyl-CAC tRNA. A wide taxonomical distribution of the gene within coryneform bacteria has been demonstrated by PCR amplification. The structural gene from linocin M18 is present at least in three Brevibacterium species, five Arthrobacter species, and five Corynebacterium species.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.     

Selective Axonal Argentaffin Staining in Rat Central Nervous System after Protein Mercuration

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.