A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

One of the two trout proopiomelanocortin messenger RNAs potentially encodes new peptides.

POMC is the precursor for a number of biologically active peptides such as ACTH, alpha-MSH, beta-MSH, and beta-endorphin. It is well known that some of these peptides, especially beta-endorphin, are involved in the regulation of reproductive functions in mammals. In order to investigate the possible role of POMC-derived peptides in the control of fish reproduction, we have cloned and sequenced two different trout POMC cDNAs called POMC A and POMC B. These cDNAs exhibited limited sequence homology (44%). The deduced amino acid sequences also showed weak similarity (43%), despite the high conservation of some peptide sequences (alpha-MSH, beta-MSH, and beta-endorphin). The POMC A coding sequence exhibited an unusual length, generating the longest endorphin ever sequenced. The long carboxy-terminal part of the beta-endorphin A contained three potential dibasic cleavage sites, allowing the occurrence of three new peptides: EQWGREEGEE, ALGE, and YHFQG. Using in situ hybridization, we found that the two POMC genes were expressed in the same pituitary cells. POMC A mRNA was the only one detectable in the hypothalamus of sexually inactive fish, whereas the two POMC genes were expressed in the hypothalamus of sexually active fish. These results indicate that two functional POMC genes are present in the rainbow trout. In POMC neurons, the expression of the POMC B gene is likely to be under the control of sexual steroids.     

The human genes for the α and γ subunits of the mast cell receptor for immunoglobulin E are located on human chromosome band 1823

The receptor with high affinity for immunoglobulin E (FcERI) is a key molecule in triggering the allergic reaction. It is tetrameric complex of one subunit, one subunit, and two disulfide-linked subunits. This receptor is present exclusively on mast cells and basophils. Molecules identical to the subunit of FcRI also form cell surface complex with other Fc receptors such as mouse FcRIIa in macrophages and most probably with human FcRIII (CD16) in natural killer (NK) cells. Here we show by in situ hybridization that the human genes for the (FCER1A) and subunits (FCER1 G) of FcERI and the gene for FcRIII (FCGR3, CD16) are located on human chromosome band 1823.     

Chromosomal localization of the human histamine H1-receptor gene

We have assigned the human histamine H₁-receptor gene to chromosome 3 by Southern blot analysis of a chromosome mapping panel constructed from humanhamster somatic cell hybrids. This assignment was confirmed by in situ hybridization on metaphase chromosomes and involved bands 3p14–p21.     

Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Membrane cholesterol content modulates ClC-2 gating and sensitivity to oxidative stress

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.     

New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria

A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.     

Lipase-catalyzed synthesis of a pure macrocyclic polyester from dimethyl terephthalate and diethylene glycol

The polymerization of dimethyl terephthalate and diethylene glycol by enzymic catalysis in toluene is described. The potential pi-pi interactions of the aromatic rings together with the relative flexibility of the diol segment have been regarded as synergy factors responsible for the formation of a pure cyclic compound. The so-called C2 macrocycle was characterized by (1)H and (13)C NMR, size-exclusion chromatography, differential scanning calorimetry, mass spectrometry, and X-ray diffraction on powder. Structurally speaking, it is made of two repeating units. The substitution of the central oxygen atom of the diol by -CH(2)- or -S- as well as the presence of ortho-substituents (-NH(2) or -NO(2)) on the phthalate were expected to disrupt the stacking of the phenyl groups more or less.