Natural substitutions at highly conserved T1-domain residues perturb processing and functional expression of squid Kv1 channels.

Shaker-type K-channel alpha-subunits (SqKv1A, B, D) expressed in neurons of the squid stellate ganglion differ in the length of their N-termini and in the species of amino acid present at several points in the T1 domain, an intracellular region involved in the tetramerization process during channel assembly. Heterologous expression of wild-type SqKv1A, B, and D in Xenopus oocytes reveals large differences in the level of both functional channels (assayed by whole-oocyte voltage clamp) and total channel protein (assayed by immunoblotting). Functional expression is poorest with SqKv1A and by far the best with SqKv1D. Biophysical properties of the three SqKv1 channels are essentially identical (assayed by cell-attached patch clamp). Site-directed mutagenesis was used to determine whether the observed differences in expression level are impacted by two residues in the T1 domain at which SqKv1A and B (but not D) differ from the consensus sequences found in many other taxa. In SqKv1A, glycine is substituted for arginine in an otherwise universally conserved sequence (FFDR in the T1(B) subdomain). In SqKv1B, glycine replaces serine in a sequence that is conserved within the Kv1 subfamily (SGLR in the T1(A) subdomain). Restoration of the consensus amino acid at these positions largely accounts for the observed differences in expression level. Analysis of the glycosylation state of aberrant versus restored alpha-subunits suggests that the anomalous amino acids in SqKv1A and B exert their influence during early steps in channel processing and assembly which take place in the endoplasmic reticulum (ER).

A family of delayed rectifier Kv1 cDNAs showing cell type-specific expression in the squid stellate ganglion/giant fiber lobe complex.

Squid giant axons are formed by giant fiber lobe (GFL) neurons of the stellate ganglion (SG). Other large motoneurons in the SG form a parallel system. A small family of cDNAs (SqKv1A-D) encoding Kv1 alpha-subunits was identified in a squid (Loligo opalescens) SG/GFL library. Members have distinct 5' untranslated regions (UTRs) and initial coding regions, but beyond a certain point (nucleotide 34 of SqKv1A) only nine differences exist. 3' UTRs are identical. Predicted alpha-subunits are nearly identical, and only the N termini differ significantly, primarily in length. RNase protection assays that use RNA isolated from specific SG regions show that SqKv1A mRNA is expressed prominently in the GFL but not in the SG proper. SqKv1B yields the opposite pattern. SqKv1D also is expressed only in the SG. SqKv1C expression was not detectable. In situ hybridizations confirm these results and reveal that SqKv1B mRNA is abundant in many large neurons of the SG, whereas SqKv1D expression is limited to small isolated clusters of neurons. SqKv1A and B are thus the predominant Kv1 mRNAs in the SG/GFL complex. Activation properties of SqKv1A and B channels expressed in oocytes are very similar to one another and compare favorably with properties of native delayed rectifier channels in GFL neurons and large SG neurons. The Kv1 complement in these squid neurons thus seems to be relatively simple. Several differences exist between cloned and native channels, however, and may reflect differences in the cellular environments of oocytes and neurons.

Knowledge, attitude, and preventive practice survey regarding AIDS comparing registered to freelance commercial sex workers in Iloilo City, Philippines.

A survey of female commercial sex workers (CSW) in Iloilo City, Philipines, was conducted in October and November 1995 to determine the level of knowledge, attitudes, and preventive practices regarding HIV/AIDS to guide future education programs. CSWs in the Philippines were categorized as registered or freelance. Registered CSWs included "hospitality girls" from licensed bars, night clubs, and massage parlors who have registered with the local social hygiene clinic (SHC). Freelance CSWs are not registered. 110 registered and 46 freelance CSWs were surveyed. We compared demographic data, scores from a basic knowledge test, and preventive practices between registered and freelance CSWs. Demographic data indicate that registered CSWs often originate from provinces outside of the Visayan Islands (25%) and most have never been married (93%). Freelance CSWs included more married (11%) and separated (11%) women from nearby cities. Knowledge test scores of registered and freelance CSWs were not significantly different. 90-96% of CSWs correctly answered questions regarding modes of transmission. However, 25% still believed it is possible to contract AIDS from using a public restroom. Registered and freelance CSWs believed their risks for AIDS to be equally great. However, 38% of freelance CSWs admit to never or almost never using condoms compared to 15% of registered CSWs. Licensed establishments and a support staff at the social hygiene clinic may provide a relatively structured working environment, giving registered CSWs security and confidence to insist on condom use. In most cases, condom use seems to depend on male customer compliance, and CSWs, especially freelancers, cannot afford to insist on condom use. The CSWs indicated that they learned most about AIDS through health personnel and television.

Tissue distribution and subcellular localization of Na+ channel mRNA in the nervous system of the squid, Loligo opalescens.

Recent cloning of a putative Na+ channel alpha subunit cDNA, GFLN1, from the squid stellate ganglion has allowed us to study the expression of this ion channel at a cellular level. In situ hybridizations with a probe derived from and specific to 3' untranslated and coding sequence of GFLN1 were used to determine its tissue distribution as well as its subcellular localization. In sections of the stellate ganglion, the probe labeled all of the cells in the giant fiber lobe (GFL) and most cells in the cellular layer of the main ganglion. In these non-GFL portions of the stellate ganglion, labeling was particularly intense in the ventral large cells and weak or absent in the dorsal small cells. In the optic lobe, only a select group of cells, the second-order visual giant neurons, were intensely labeled. These results are consistent with electrophysiological data that show GFL-like Na+ currents in rare large cells dissociated from the optic lobe and in most but not all cells from the non-GFL part of the stellate ganglion. In sections of the subesophageal mass of the central nervous system, strong labeling for GFLN1 mRNA occurred in the fin lobe, posterior chromatophore lobe, central and latero-ventral palliovisceral lobes, and posterior pedal lobe. In all cases, labeling was detected only in the cellular layer of these tissues and never in nerves or neuropil. In situ hybridization with dissociated GFL neurons maintained in primary culture verified that Na+ channel mRNA is confined to the cell body. These results indicate that GFLN1 is expressed predominately in large cells with large or long axons, and that this mRNA is restricted to the cell bodies of these neurons.

[Synthesis of prolactin by human decidua in vitro (author's transl)].

Human decidua, chorion, amnion and placenta from 1st., 2nd. and 3rd. trimesters of gestation were investigated for synthesis and secretion of prolactin by in vitro incubation of these tissue fragments in medium 199. Prolactin content in decidua was found to be significantly higher than those in chorion, amnion or placenta at any stages of gestation. During 6 hours of incubation, decidua secreted significantly more prolactin into medium than did chorion, amnion or placenta. Amount of prolactin secreted by decidua was significantly higher than prolactin content in tissue before incubation. Decidua were also incubated in medium 199 with or without actinomycin-D (20-200 microgram/ml), puromycin (200 microgram/ml) or cycloheximide (100-200 microgram/ml) for 12 hours. Both total prolactin secretion into medium and prolactin content in tissue after incubation were significantly lower as compared to control without inhibitor. Decidua of 2nd. trimester of gestation was noted to secrete more prolactin into medium than decidua of 1st. or 3rd. trimester of gestation. In further studies, radioleucine (200 uCi) was incubated for 12 hours with decidua (10 grams) from 2nd. trimester of gestation, the incorporated radioprotein in medium or tissue was extracted, fractionated by Sephadex G-100 chromatography and by 10% SDS-polyacrylamide gel electrophoresis. Peaks of radioactivity and immunoreactive prolactin in gel slices were coincident with the peak of human pituitary prolactin (NIAMDD, hPRL, Friesen No. 1) at Kf = 0.62 (gel slice No. 31). In addition, mRNA was extracted from decidual tissue by oligo-d (T)-cellulose chromatography, which was then used for cell-free system translation study. The translation product of decidual mRNA and rabbit reticulocyte lysate was shown to be identical to human pituitary prolactin by analysis of gel electrophoresis. These results demonstrate that prolactin synthesized and secreted by human decidua is identical to human pituitary prolactin. The synthetic activity is most prominent in the decidua of 2nd. trimester of gestation.

Synthesis of prolactin by human decidua in vitro.

Human decidua, chorion, amnion and placenta from the 1st., 2nd. and 3rd. trimesters of gestation were investigated for synthesis and secretion of prolactin by in vitro incubation of these tissue fragments in medium 199. Prolactin content in decidua was found to be significantly higher than that in chorion, amnion or placenta at any stages of gestation. During 6 hours of incubation, decidua secreted significantly more prolactin into medium than did chorion, amnion or placenta. The amount of prolactin secreted by decidua was significantly higher than the prolactin content in tissue before incubation. Decidua were also incubated in medium 199 with or without actinomycin-D (20-200 micrograms/ml), puromycin (200 micrograms/ml) or cycloheximide (100-200 micrograms/ml) for 12 hours. Both total prolactin secreted into medium and prolactin content in tissue after incubation were significantly lower than the control without inhibitor. Decidua of 2nd. trimester of gestation was noted to secrete more prolactin into medium than decidua of 1st. or 3rd. trimester of gestation. In further studies, 3H-leucine (200 muCi) was incubated for 12 hours with decidua (10 grams) from 2nd. trimester of gestation. The incorporated protein in medium or tissue was extracted, fractionated by gel filtration through Sephadex G-100 column and by 10% SDS-polyacrylamide gel electrophoresis. Peaks of 3H count and immunoreactive prolactin in gel slices were coincident with the peak of standard pituitary prolactin at gel slices' No. 31 (Rf = 0.62). These results demonstrate that prolactin is synthesized and secreted by human decidua, which is identical to human pituitary prolactin. The synthetic activity is most prominent in the decidua of the 2nd. trimester of pregnancy.