Assignment of the αB-crystallin gene to human chromosome 11

Using a human αB-crystallin genomic probe and human-mouse somatic cell hybrids, the human αB-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3–23.1.     

Pheromone-mediated sexual selection in the mothUtetheisa ornatrix: Olfactory receptor neurons responsive to a male-produced pheromone

Male Utetheisa ornatrixhave a pair of eversible glandular brushes (coremata) which are displayed during precopulatory interactions with the female. Earlier studies have shown that a pheromone associated with the coremata, hydroxydanaidal (HD), is derived by the males from pyrrolizidine alkaloids (PAs) that they sequester as larvae from their foodplants (Crotalariaspp.) The PAs impart a distastefulness upon Utetheisathat protects both larvae and adults against predation. The receptor neurons specialized for detection of HD are housed in sensilla whose morphological features, as revealed by scanning electron microscopy, classify them as sensilla basiconica. The sensitivity and dynamic range of these receptor neurons were largely unaffected by whether the females were raised on an alkaloid-free diet or on a diet supplemented with Crotalariaseeds. Acetylation of the hydroxyl group of HD substantially reduced the activity of the molecule. None of the antennal sensilla examined contained receptor neurons sensitive to a PA (monocrotaline) or its N-oxide.     

Determination of the concentration of complex boronated compounds in biological tissues by inductively coupled plasma atomic emission spectrometry

The application of inductively coupled plasma atomic emission spectrometry (ICP-AES) to the determination of the concentration of complex boron-containing compounds in biological tissue samples is described. Tissue digestion is achieved with perchloric acid and hydrogen peroxide in 1 hr at 75 degrees C. The ICP-AES method gave a linear response for elemental boron concentration in the range 0.05 to 100 ppm and does not require the reduction of the boron to a simple species, such as boric acid. Complete recovery of boron in complex boron cluster compounds was obtained. The procedure has been applied to the determination of the boron content in compounds synthesised for neutron capture therapy and is suitable for use in biodistribution studies of such compounds.     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA     

DNA synthesis in Langerhans'cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

Abstract The proportion of Langerhans'cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [³H]TdR autoradiography with enzyme histochemistry. After injection of [³H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (p± SE mean) for cytospins, 40 min after injecting [³H]TdR, was 1.6 ± 0.15%, doubling to 3–4% from 7–17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans'cells rising from 18% at 40 min after injection, to approximately 45%, on days 1–4 after injection. These results suggest that the proliferation of Langerhans'cells in normal mouse skin might be higher than was previously thought to be the case.     

Prostaglandin E2 enhances the sodium conductance of exocrine glands in isolated frog skin (Rana esculenta)

Summary Prostaglandins are known to stimulate the active transepithelial Na⁺ uptake and the active secretion of Cl^– from the glands of isolated frog skin. In the present work the effect of prostaglandin E₂ (PGE₂) on the glandular Na⁺ conductance was examined. In order to avoid interference from the Na⁺ uptake and the glandular Cl^– secretion the experiments were carried out on skins where the Cl^– secretion was inhibited (the skins were bathed in Cl^– Ringer's solution in the presence of furosemide, or in NO ₃ ^– Ringer's solution), and the active Na⁺ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at –100 mV, basolateral solution was taken as reference). When PGE₂, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na⁺ efflux. Together with the increase in Na⁺ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na⁺, and when one Na⁺ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na⁺ is confined to the exocrine glands.     

Role of the Blood-Brain Barrier in the Formation of Long-Chain ω-3 and ω-6 Fatty Acids from Essential Fatty Acid Precursors

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.     

Biexponential Kinetics of (R)-α-[3H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor

The H3 histamine receptor is a high-affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization-induced histamine release. We have used (R)-alpha-[3H]methylhistamine, a specific, high-affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD = 0.68 nM; Bmax = 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: N alpha-methylhistamine, (R)-alpha-methylhistamine greater than histamine, thioperamide greater than impromidine greater than burimamide greater than dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on-and-off rates. Density of (R)-alpha-[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex greater than hypothalamus greater than brainstem greater than cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undifferentiated.     

N-Methyl-D-Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site

In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.