Role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission.

Herpes simplex virus (HSV) glycoprotein D (gD) is essential for virus entry into cells, is modified with mannose-6-phosphate (M-6-P), and binds to both the 275-kDa M-6-P receptor (MPR) and the 46-kDa MPR (C. R. Brunetti, R. L. Burke, S. Kornfeld, W. Gregory, K. S. Dingwell, F. Masiarz, and D. C. Johnson, J. Biol. Chem. 269:17067-17074, 1994). Since MPRs are found on the surfaces of mammalian cells, we tested the hypothesis that MPRs could serve as receptors for HSV during virus entry into cells. A soluble form of the 275-kDa MPR, derived from fetal bovine serum, inhibited HSV plaques on monkey Vero cells, as did polyclonal rabbit anti-MPR antibodies. In addition, the number and size of HSV plaques were reduced when cells were treated with bovine serum albumin conjugated with pentamannose-phosphate (PM-PO4-BSA), a bulky ligand which can serve as a high-affinity ligand for MPRs. These data imply that HSV can use MPRs to enter cells; however, other molecules must also serve as receptors for HSV because a reasonable fraction of virus could enter cells treated with even the highest concentrations of these inhibitors. Consistent with the possibility that there are other receptors, HSV produced the same number of plaques on MPR-deficient mouse fibroblasts as were produced on normal mouse fibroblasts, but there was no inhibition with PM-PO4-BSA with either of these embryonic mouse cells. Together, these results demonstrate that HSV does not rely solely on MPRs to enter cells, although MPRs apparently play some role in virus entry into some cell types and, perhaps, act as one of a number of cell surface molecules that can facilitate entry. We also found that HSV produced small plaques on human fibroblasts derived from patients with pseudo-Hurler's polydystrophy, cells in which glycoproteins are not modified with M-6-P residues and yet production of infectious HSV particles was not altered in the pseudo-Hurler cells. In addition, HSV plaque size was reduced by PM-PO4-BSA; therefore, it appears that M-6-P residues and MPRs are required for efficient transmission of HSV between cells, a process which differs in some respects from entry of exogenous virus particles.     

Charge displacement in bacteriorhodopsin during the forward and reverse bR-K phototransition.

Dried oriented purple membrane samples of Halobacterium salinarium were excited by 150 fs laser pulses of 620 nm with a 7 kHz repetition rate. An unusual complex picosecond electric response signal consisting of a positive and a negative peak was detected by a sampling oscilloscope. The ratio of the two peaks was changed by 1) reducing the repetition rate, 2) varying the intensity of the excitation beam, and 3) applying background illumination by light of 647 nm or 511 nm. All of these features can be explained by the simultaneous excitation of the bacteriorhodopsin ground form and the K intermediate. The latter was populated by the (quasi)continuous excitation attributable to its prolonged lifetime in a dehydrated state. Least-square analysis resulted in a 5 ps upper and 2.5 ps lower limit for the time constant of the charge displacement process, corresponding to the forward reaction. That is in good agreement with the formation time of K. The charge separation driven by the reverse phototransition was faster, having a time constant of a 3.5 ps upper limit. The difference in the rates indicates the existence of different routes for the forward and the reverse photoreactions.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

A flavodoxin that is required for enzyme activation: the structure of oxidized flavodoxin from Escherichia coli at 1.8 A resolution.

In Escherichia coli, flavodoxin is the physiological electron donor for the reductive activation of the enzymes pyruvate formate-lyase, anaerobic ribonucleotide reductase, and B12-dependent methionine synthase. As a basis for studies of the interactions of flavodoxin with methionine synthase, crystal structures of orthorhombic and trigonal forms of oxidized recombinant flavodoxin from E. coli have been determined. The orthorhombic form (space group P2(1)2(1)2(1), a = 126.4, b = 41.10, c = 69.15 A, with two molecules per asymmetric unit) was solved initially by molecular replacement at a resolution of 3.0 A, using coordinates from the structure of the flavodoxin from Synechococcus PCC 7942 (Anacystis nidulans). Data extending to 1.8-A resolution were collected at 140 K and the structure was refined to an Rwork of 0.196 and an Rfree of 0.250 for reflections with I > 0. The final model contains 3,224 non-hydrogen atoms per asymmetric unit, including 62 flavin mononucleotide (FMN) atoms, 354 water molecules, four calcium ions, four sodium ions, two chloride ions, and two Bis-Tris buffer molecules. The structure of the protein in the trigonal form (space group P312, a = 78.83, c = 52.07 A) was solved by molecular replacement using the coordinates from the orthorhombic structure, and was refined with all data from 10.0 to 2.6 A (R = 0.191; Rfree = 0.249). The sequence Tyr 58-Tyr 59, in a bend near the FMN, has so far been found only in the flavodoxins from E. coli and Haemophilus influenzae, and may be important in interactions of flavodoxin with its partners in activation reactions. The tyrosine residues in this bend are influenced by intermolecular contacts and adopt different orientations in the two crystal forms. Structural comparisons with flavodoxins from Synechococcus PCC 7942 and Anaebaena PCC 7120 suggest other residues that may also be critical for recognition by methionine synthase.     

Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.     

Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly

Two related transmembrane proteins in mammalian cells bind the mannose 6-phosphate recognition marker present on all soluble lysosomal hydrolases. Recent studies of cells that express only one or neither of these proteins have shed light not only on their function in directing lysosomal enzymes into the endocytic pathway but also on their critical role in transport vesicle formation in the trans Golgi network. One of these proteins also binds insulin-like growth factor II (IGF-II) and may be an important part of the IGF-dependent system that regulates development.     

A broadly active viral inhibitor in human and animal organ extracts and body fluids

To help assess the possibility that a newly described viral inhibitor from cell cultures might play a natural defensive role in vivo, its distribution and concentration in human and animal organ extracts and body fluids were investigated. The concentration of the inhibitor was high in human liver, heart muscle, splenic extracts, and human serum and milk. The inhibitor in the body was indistinguishable from a previously described inhibitor produced in cell cultures that was characterized by broad antiviral activity, lack of target cell species specificity, lack of induction of stable antiviral activity in cells, rapid reversibility of antiviral action, prevention of virus attachment, and stability at 100 degrees C. Sixteen virus plaque reduction units of the inhibitor diminished the yield of poliovirus in vitro by more than 1000-fold. Additional evidence that contact-blocking viral inhibitor (CVI) inhibits vaccinia virus attachment to cells is presented. A role for the inhibitor in natural defense against viral infections is possible.     

Isolation of taste buds from the foliate papillae of the rabbit

Summary A method to isolate taste buds from the foliate papillae of the rabbit tongue is described. The method comprises (a) separation of the epidermis from the dermal layer after treatment with dilute acetic acid, and (b) mechanical removal of the taste buds from the epithelium with the use of a surgical needle. The procedure yields taste buds that are morphologically well preserved, and in quantities sufficient to enable a detailed biochemical characterization. Preliminary tests have shown the taste buds to have biochemical properties clearly distinct from those of the adjacent epithelium. The method may provide a basis for studying the molecular mechanism of taste perception in greater detail.On leave of absence from the Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.