The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans-Golgi network of HeLa M-cells, but a proportion was also localized to multivesicular bodies (MVBs). Depletion of NHE8 in HeLa M-cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes, and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVBs. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVBs.     

An active site-tyrosine-containing heptapeptide from D-amino acid oxidase

The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.     

Ethanenitronate is a peroxide-dependent suicide substrate for catalase

We find ethanenitronate (formula; see text) to be a H2O2- (and peracetic acid-) dependent suicide substrate for bovine liver catalase (E) which converts E to Em, a modified form of the enzyme. The catalytic and suicide pathways are related to E, Em, Compound I, and Compound II according to the following scheme. (formula; see text) The catalytic cycle generates free radical products (EN.) which then participate in an O2-dependent chain reaction. Within experimental error the exclusive target for inactivation by EN- is Compound II. This partitions in the ratio (k4 = 1.2 M-1 s-1)/(k3 = 1.6 M-1 s-1) to generate Em and E, respectively. The species Em acquires 1 eq of 14C/ferriheme from [1-14C]ethanenitronate which is firmly (presumably covalently) affixed to the protein moiety. According to the standard H2O2 assay, Em is 7% as active catalytically as E. We regard inactivation as resulting from that fraction of EN. in the E...EN. complex which fails to diffuse from the complex because it is trapped by reaction with a neighboring amino acid residue to generate Em irreversibly. (formula; see text) This mechanism is identical to that deduced previously for suicide inactivation of horseradish peroxidase by alkane nitronates (Porter, D. J. T., and Bright, H. J. (1983) J. Biol. Chem. 258, 9913-9924) with the exception that EN. is trapped in that case by a methine carbon at the edge of the ferriheme rather than by the apoenzyme. The labeled residue in the catalase apoenzyme probably resides at or near the site of reduction of Compound II.     

The cyanogenic substrate for horseradish peroxidase is a conjugated enamine

We identify the cyanogenic substrate for horseradish peroxidase (HRP) as a conjugated enamine and explore this unusual reaction using alpha-aminocinnamate (RH) as follows. 1) HRP catalyzes the oxidation of RH by O2 (and its peroxidation by H2O2 to form R-R) to produce, simultaneously, CN- and benzaldehyde cyanohydrin. 2) RH is transient and must be generated in situ. The properties of the cyanogenic reaction of HRP are independent of the method of preparation of RH (whether this be condensation of NH3 with phenylpyruvate, enzymatic hydrolysis of glycyldehydrophenylalanine, or oxidation of L-phenylalanine by L-amino acid oxidase). 3) The oxidation of RH is a free radical chain reaction initiated by HRP Compounds I and II (I (or II) + RH----R. + II (or HRP], propagated by RO2. (R. + O2----RO2., RO2. + RH----R. + RO2H), and terminated by recombination reactions such as 2R.----R2 and RO2.----R' + HO2. followed by R. + HO2.----RH + O2. KMnO4 and K3Fe(CN)6 can substitute for HRP. 4) The proximal precursor of CN- and cyanohydrin is postulated to be RO2H (phi-CH(-O2H)-CCO2-(= NH]. These results explain why cyanide is generated from the synergistic action of HRP and L-amino acid oxidase on aromatic L-amino acids and O2 and suggest that the requirement for a beta-aryl substituent on the enamine originates in the reaction of RH with HRP, or of R with O2, rather than the imine/enamine tautomerization of the L-amino acid oxidase product.     

Evaluation of algorithms for ratio imaging in fluorescence microscopy

Ratio imaging in fluorescence microscopy is used in measuring parameters such as pH, pCa, cytoplasmic porosity, and the relative concentration of fluorescent analogs within single cells. The fastest method for ratio imaging is to use lookup tables on special-purpose image processors. Since lookup tables store integers in integer addresses, using a lookup table will generate rounding errors. The magnitude of the error will depend on the transformation performed and on the number of levels used in the lookup table. We examined ratio imaging by lookup table and computed the errors generated by both inversion and log subtraction methods. Both uniformly fluorescing fields and fluorescing cell images were employed to provide data for use in confirming our calculations and illustrating both the magnitude and spatial incidence of errors. It is shown that, through proper design of lookup tables, a significant reduction can be made in the errors generated in comparison with common methods available in most image processors.     

Idiotype network components are involved in the murine immune response to simian virus 40 large tumor antigen

Summary Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag), a monoclonal antibody specific for SV40 T-Ag (Ab-1 preparation), and a monoclonal anti-idiotypic antibody (anti-Id), designated 58D, were used to analyze the humoral immune response of Balb/c mice either immunized with recombinant SV40 T-Ag or challenged with SV40-transformed cells. Inhibition assays indicated that antibodies from mice immunized with SV40 T-Ag and from those bearing SV40 tumor inhibited the SV40 T-Ag/Ab-1 reaction. These data suggested that the antibody response in immunized or tumorchallenged mice recognized similar epitope(s) on SV40 T-Ag to that detected by the monoclonal Ab-1. These anti-(SV40 T-Ag) response antibodies also inhibited the Ab-1/anti-Id reaction and recognized the anti-Id in direct binding assays. Together, these data indicate that murine anti-(SV40 T-Ag) responses shared an idiotope with a monoclonal anti-(SV40 T-Ag) Ab-1 preparation. This idiotope, which is recognized by the monoclonal anti-Id preparation, 58D, appears to be involved in the humoral immune response to SV40 T-Ag in both SV40-T-Ag-immunized and tumor-bearing mice. The monoclonal anti-Id preparation may represent a focal point for manipulating the humoral immune response to tumors induced by SV40-transformed cells.     

The effect of sulfide and an increased food supply on the meiofauna and macrofauna at the East Flower Garden brine seep

A sulfurous brine seep at the East Flower Garden Bank, northwest Gulf of Mexico, produces conditions conducive to the growth of a luxuriant prokaryotic biota. Hydrodynamic cropping continually harvests this biota and distributes it to sandy-bottom and hard-bank benthic communities downstream of the seep. Consequently, both macro- and meiofaunal abundances are dramatically increased above the regional norm in parts of the seep system. When sulfide is present, the lower Bilaterian groups belonging to the meiofauna dominate the community; without sulfide, macrofaunal groups, particularly crustaceans, dominate the community. Outside the influence of the seep, meiofaunal copepods predominate. Changes in taxonomic composition and abundance indicate that the sandy-bottom benthos at 70–80 m depth at the East Flower Garden bank is foodlimited and that, under these conditions, meiofauna, particularly the higher Bilaterian groups, dominate the community numerically. Perhaps, under food-limiting conditions, meiofauna compete favorably with macrofauna for food.     

Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.