Cloning of cDNAs encoding human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Comparison of their deduced amino acid sequences

We have isolated and sequenced cDNA clones corresponding to the entire coding sequences of the human lysosomal membrane glycoproteins, lamp-1 and lamp-2 (h-lamp-1 and h-lamp-2). The deduced amino acid sequences indicate that h-lamp-1 and h-lamp-2 consist of 416 and 408 amino acid residues, respectively, and suggest that 27 and 28 NH2-terminal residues are cleavable signal peptides. The major portions of both h-lamp-1 and h-lamp-2 reside on the luminal side of the lysosome and are heavily glycosylated by N-glycans: h-lamp-1 and h-lamp-2 were found to contain 19 and 16 potential N-glycosylation sites, respectively. The findings are consistent with the results obtained by endo-beta-N-acetylglucosaminidase F treatment of h-lamp-1 and h-lamp-2 precursors, described in the preceding paper (Carlsson, S. R., Roth, J., Piller, F., and Fukuda, M. (1988) J. Biol. Chem. 263, 18911-18919). These N-glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and serine in h-lamp-1 or proline and threonine in h-lamp-2. The two domains of h-lamp-1 on each side of the hinge region are homologous to each other, whereas no such homology was detected between the two domains of h-lamp-2. Both proteins have one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH terminus, and contain a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end. Comparison of h-lamp-1 and h-lamp-2 sequences reveal strong homology between the two molecules, particularly in the proximity to the COOH-terminal end. It is possible that this portion is important for targeting the molecules to lysosomes. These results also suggest that lamp-1 and lamp-2 are evolutionarily related. Comparison of known lamp-1 sequences among different species, on the other hand, show that human lamp-1 has more similarity to lamp-1 from other species than to human lamp-2. This fact, taken together with the finding that h-lamp-2 lacks repeating domains, suggests that lamp-1 and lamp-2 diverged from a putative ancestor gene in early stages of evolution. These results also suggest that lamp-1 and lamp-2 probably have distinctly separate functions despite the fact that they share many structural features.     

Na channels and two types of Ca channels in rat pancreatic B cells identified with the reverse hemolytic plaque assay

The reverse hemolytic plaque assay (RHPA) was used to study the secretory properties of single rat pancreatic B cells, and to identify insulin-secreting cells for patch-clamp experiments. In secretion studies using the RHPA, we find that the percentage of secreting B cells and the amount of insulin secreted per B cell increase as the glucose concentration is raised from 0 to 20 mM. Using the whole-cell variation of the patch-clamp technique, we find that identified B cells have three types of channels capable of carrying inward current: (a) tetrodotoxin-sensitive, voltage-dependent Na channels, which are nearly completely inactivated at -40 mV, (b) fast deactivating (FD) Ca channels, and (c) slowly deactivating (SD) Ca channels. We have shown that Na channels are functionally significant to the B cell, because tetrodotoxin partially inhibits glucose-induced insulin secretion. The properties of FD and SD Ca channels differ in several respects. FD channels deactivate at -80 mV, with a time constant of 129 microseconds, they are half-maximally activated near +15 mV, they do not inactivate during 100 ms, they conduct Ba2+ better than Ca2+, and they are very sensitive to washout during intracellular dialysis. SD channels, on the other hand, deactivate with a time constant of 2.8 ms, they are half-maximally activated near -5 mV, they inactivate rapidly, they conduct Ba2+ and Ca2+ equally well, and they are insensitive to washout.     

Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence.

In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet. Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA. The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes. The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency. On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons. Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs.     

Properties of two types of calcium channels in clonal pituitary cells

The calcium currents of GH3 cells have been studied using the whole cell variant of the patch-clamp technique. Under conditions that eliminate sodium and potassium currents, we observed inward currents that activated within a few milliseconds, and deactivated with two time constants, approximately 150 microseconds and 3 ms at -80 mV, 18-20 degrees C. The components are called FD and SD (fast deactivating and slow deactivating). Both components are calcium currents, and are greatly reduced when magnesium is substituted for most of the calcium in the bath. In addition to (a) their different rates of deactivation, the two components differ in a number of other properties. (b) The SD component inactivates almost completely, with a time constant of 23 ms at 20 mV, 19 degrees C. The FD component, on the other hand, shows little or no sign of inactivation, and is almost the same in amplitude from 10 to 100 ms. The components thus seem quite independent of each other, and must arise from two independent sets of channels. (c) The FD channels activate more rapidly than SD at 20 mV, by a factor of approximately 2 as is shown in several ways. (d) In 10 Ca or 10 Ba, the activation curve for SD channels is approximately 20 mV more negative than for FD or Na channels. (e) FD channels conduct barium ions more effectively than calcium by a ratio of approximately 2. (f) FD channels "wash out" within minutes after the patch electrode breaks into a cell, whereas SD channel current remains relatively stable. It is argued that SD channels, because of their negative activation threshold, are involved in electrical events near threshold, and that FD channels are best suited for calcium injection once a spike has been initiated.     

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA

P450_scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450_scc indicates P450_scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the $\text{PstI}$ site of pBR322 by dC·dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450_scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.