Litter in a first–order stream of a temperate deciduous forest (Margaraça Forest, central Portugal)

To evaluate the importance and fate of organic matter inputs in forested streams, we determined the litterfall inputs and the benthic coarse particulate organic matter (CPOM) in one headwater stream flowing through a mixed deciduous forest, during one year. Both vertical traps and the stream bottom were sampled monthly. The material collected was sorted into four main categories: leaves, fruits and flowers, twigs and debris. Litter production was 715 g m−2 y−1 and seasonal, with 73% of the annual total during October–December (autumn). Leaves comprised the largest litter component. Benthic organic matter was 1880 g m−2 y−1, and was also seasonal. Highest accumulation was attained in spring, and twigs and branches comprised the major component. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human Immunodeficiency Virus (HIV) Envelope Binds to CXCR4 Independently of CD4, and Binding Can Be Enhanced by Interaction with Soluble CD4 or by HIV Envelope Deglycosylation

Chemokine receptor CXCR4 (also known as LESTR and fusin) has been shown to function as a coreceptor for T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have developed a binding assay to show that HIV envelope (Env) can interact with CXCR4 independently of CD4 but that this binding is markedly enhanced by the previous interaction of Env with soluble CD4. We also show that nonglycosylated HIV-1_SF-2 gp120 or sodium metaperiodate-treated oligomeric gp160 from HIV-1₄₅₁ bound much more readily to CXCR4 than their counterparts with intact carbohydrate residues did.In the recent past, several members of the family of chemokine receptors have been identified as cofactors for human immunodeficiency virus type 1 (HIV-1) entry (1, 6, 8, 10). Specifically, CCR5 (as well as CCR3 and CCR2b in some instances) has been shown to mediate entry of viruses characterized as macrophage tropic or dual tropic (1, 5–8), while CXCR4 has been shown to mediate entry of T-cell-tropic or dual-tropic strains (7, 10). While several ligands have been found for CCR5, CXC chemokine stromal derivative factor (SDF1) remains the only known ligand for CXCR4 (4, 24). Coimmunoprecipitation studies have shown that HIV-1 Env from T-cell-tropic strains forms a complex with CD4 and CXCR4 (18), but the nature of the binding events leading to the formation of this complex and the possibility of a direct interaction between HIV Env and CXCR4 remained speculative. Data from Hesselgesser et al. (15) have more recently shown that gp120 from the T-cell-tropic strains IIIB or BRU was able to compete with SDF1 for binding to CXCR4 in hNT cells (a neuronal CD4-negative cell line), indicating the possibility of a direct interaction between CXCR4 and gp120, but no information was presented on the relevance of the interaction with CD4. Other data have shown that gp120 from macrophage-tropic strains of HIV might be able to bind directly to CCR5 and that the affinity for binding between the two molecules can be increased significantly by the presence of soluble CD4 (sCD4) (34), although this effect could not be reproduced by a different group (32).We have performed the following studies to determine if HIV Env binds to CXCR4 independently of CD4 and, if so, what would be the effect of previous binding of HIV Env to sCD4.

CD4-independent binding of HIV Env to CXCR4.

The phenotypes of the T-cell lines CEM-SS and Jurkat 25 (J25) were evaluated with respect to surface expression of both CD4 and CXCR4. J25 clone 22F6 cells (3, 21) were grown in complete medium (RPMI 1640, 2% penicillin-streptomycin, 2% l-glutamine; BioWhittaker, Walkersville, Md.) containing heat-inactivated 10% fetal calf serum at 37°C in a 5% CO₂ atmosphere. CEM-SS is a T-cell line that was obtained from the AIDS Research and Reference Reagent Program and maintained in complete medium. CEM-SS cells were derived from a human lymphoblastoid tumor (22, 23). Commercial monoclonal antibody (MAb) to CD4 (mouse immunoglobulin G2a [IgG2a], clone S3.5), fluorescein isothiocyanate (FITC) labeled, and the necessary isotypic controls were obtained from Caltag Laboratories (San Francisco, Calif.). Mouse MAb 12G5 against CXCR4 was raised in BALB/c mice and has been described previously (9). Goat anti-mouse IgG–FITC was purchased from Becton Dickinson (San Jose, Calif.). Flow cytometric analysis was performed on a Becton Dickinson FACScan cytometer equipped with a 15-mW argon laser emitting at 488 nm. Dead cells were detected on the basis of their scatter and eliminated from the analysis. Live cells (10,000) were analyzed for each marker. CXCR4 surface expression was determined by washing the cells taken in logarithmic growth phase with phosphate-buffered saline (PBS) containing 1% horse serum and incubating them with 10 μl of 12G5 antibody/100 μl (0.16 mg/ml) at 4°C for 30 min. The cells were then washed again in PBS, and a secondary goat anti-mouse IgG–FITC (Becton Dickinson) was incubated with the cells for another 30 min at 4°C. Finally, the cells were washed with PBS and fixed with 2% paraformaldehyde. As a control, equal amounts of mouse IgG2a (the same isotype as 12G5) were used. Both cell lines expressed significant levels of CXCR4 on their surfaces (Fig. ​(Fig.1),1), but only CEM-SS had measurable levels of surface CD4. This characteristic of the phenotype of J25 cells, with respect to CD4 expression, has been reported before (3). To assess binding of HIV Env to CXCR4, the following binding assay was developed. Oligomeric gp160 (ogp160) was purified from cell cultures (obtained from T. C. Van Cott (Henry M. Jackson Foundation, Rockville, Md.) infected with HIV₄₅₁ (17). The cells were washed once with PBS and then incubated with ogp160 for 1 h at 37°C in RPMI medium. The cells were washed again in PBS and incubated with 10 μg of human MAb 1331A [IgG3(λ)]/ml, which is specific for the C terminus of gp120 (i.e., amino acids 510 to 516 of HIV_LAI), or with a human MAb against p24 (MAb 71-31) as a control (12) for 30 min at 4°C. The secondary antibody was a goat anti-human IgG phycoerythrin labeled (Caltag). The cells were fixed in 2% paraformaldehyde, and the fluorescence intensity was determined by flow cytometry. Background was obtained by adding MAb 1331 and goat anti-human IgG, phycoerythrin labeled, to the cells in the absence of ogp160. The results of the binding assay with ogp160 from HIV₄₅₁ and both cell lines are shown in Fig. ​Fig.2A.2A. By using the high-affinity human MAb 1331A against the C-terminal region of gp120, our assay was able to detect significant binding of the ogp160 molecule to the surfaces of both cell lines even at concentrations of only 88 nM. The very high relative affinity of MAb 1331A for the gp120 molecule appears to be critical to demonstrate this interaction, as other antibodies with lower relative affinities for gp120 were incapable of detecting this low-level binding (data not shown). The binding of ogp160 to the CD4-expressing CEM-SS cells was several orders of magnitude higher than that to the J25 cells. To prove the specificity of the binding assay for CXCR4, a synthetic form of SDF1 was produced and tested for its ability to block infection by the HIV-1 strain NL_4-3 in HeLa CD4-positive long terminal repeat (LTR)-LacZ cells. These data have been published elsewhere (2). SDF1 synthesis and composition have been described previously (24). Exposure of J25 cells to SDF1 was shown to produce a dose-dependent blockage of the binding of ogp160 to the surfaces of the J25 cells (Fig. ​(Fig.2B),2B), indicating the specific nature of the assay. Open in a separate windowFIG. 1Phenotype analysis of CEM-SS and J25 cell lines. Thin solid line, background; thick solid line, CD4; dashed line, CXCR4.Open in a separate windowFIG. 2(A) Binding of ogp160 from HIV₄₅₁ to the surfaces of CEM-SS or J25 cells. Fluorescence intensity is expressed on a logarithmic scale on the x axis, with each line representing one-half log. Concentrations of ogp160 are shown at the right of each graph. The experiments were done in duplicate to ensure consistency of results. (B) Effect of RANTES (250 nM) or increasing amounts of SDF1 (up to 250 nM) on binding of ogp160 (355 nM) to J25 cells. The results are expressed as mean channel fluorescence. Experiments were repeated twice to ensure consistency of results.To further test the fact that HIV Env binding to CXCR4 could occur independently of CD4, and to evaluate the effect of prior binding of Env to sCD4, the following experiments were performed. We preexposed CEM-SS as well as J25 cells to either the anti-CD4 antibody Leu3a (Becton Dickinson), which blocks the CD4 binding domain of HIV Env, or OKT4 (Ortho Diagnostics, Costa Mesa, Calif.), which does not block binding of HIV Env to CD4. The cells were then tested for their ability to bind ogp160 to their surfaces. As shown in Fig. ​Fig.3,3, OKT4 had no significant effect on the binding of ogp160 to either CEM-SS or J25 cells while Leu3a readily inhibited binding of ogp160 to CEM-SS cells but had no such effect on J25 cells. Furthermore, when ogp160 was allowed to react in advance with recombinant sCD4 produced in CHO cells (Intracel, Issaquah, Wash.) for 30 min at 4°C at a concentration of 1 μg/ml, we were able to show a clear decrease in the surface binding of ogp160 to CEM-SS cells while the opposite, an obvious enhancement in surface binding, was demonstrated for J25 cells (Fig. ​(Fig.3).3). Open in a separate windowFIG. 3Binding of ogp160 to CEM-SS or J25 cells after exposure of the cells to the anti-CD4 antibodies Leu3a (thin solid lines), OKT4 (dotted lines), or a combination of ogp160 with sCD4 (dashed lines). The shaded areas represent background. The thick solid lines represent binding in the absence of antibodies or sCD4. The experiments were performed in quadruplicate with similar results. Mean channel fluorescence is represented on the x axis.Taken together, these data indicate that HIV Env can bind to CXCR4 independently of CD4. On the other hand, prior interaction of HIV Env with CD4 results in a clear increase in the binding of HIV Env to CXCR4.

Relevance of the glycosylation state of HIV Env in binding to CXCR4.

The binding of HIV Env to CD4 is dependent on the appropriate conformation of the Env molecule (27), which can be significantly altered by changes in its carbohydrate content. We next tested the hypothesis that alterations in the carbohydrate moieties of Env would affect its binding to CXCR4. To do so, we used the gp120 molecule from HIV_SF2, produced in CHO cells, and its counterpart, nonglycosylated HIV_SF2 Env 2-3, produced in yeast strain 2150, and tested both in the binding assay with CEM-SS or J25 cells. HIV_SF-2 gp120 and its nonglycosylated counterpart, Env 2-3, were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Kathelyn Steimer, Chiron Corp. (13, 14, 19, 26, 29–31). The results are shown in Fig. ​Fig.4.4. As expected, nonglycosylated HIV_SF2 Env 2-3 bound to the surfaces of the CEM-SS cells to a lesser extent than did HIV_SF2 gp120. On the other hand, and unexpectedly, nonglycosylated HIV_SF2 Env 2-3 bound much more readily to the surfaces of the J25 cells than its glycosylated counterpart, HIV_SF-2 gp120, even when used at equal molar concentrations. To determine whether these findings could be generalized to other Env molecules that lacked intact carbohydrate molecules, we treated ogp160 with sodium metaperiodate. ogp160 from HIV₄₅₁ at 1.25 μg/ml was treated with sodium metaperiodate (Sigma, St. Louis, Mo.) in acetate buffer for 2 h at 4°C in the dark (33). The cells to be tested had been treated previously with 1% glycine (Sigma) for 30 min at 37°C. Such treatment results in the oxidation and cleavage of the carbohydrate hydroxyl groups without affecting the structure of the polypeptide chains (33). Nonspecific binding by the resulting aldehyde groups was prevented by blocking the target cells beforehand with 1% glycine. The results are shown in Fig. ​Fig.4.4. Sodium metaperiodate treatment of ogp160 resulted in a marked inhibition of the binding of ogp160 to the surfaces of the CEM-SS cells. In contrast, sodium metaperiodate treatment of ogp160 resulted in a very clear increase in the binding of HIV Env to the surfaces of the J25 cells. The preexposure of CEM-SS cells to SDF1 did not significantly affect the binding of ogp160 or sodium metaperiodate-treated ogp160. On the other hand, preexposure of J25 cells to 250 nM SDF1 resulted in a marked decrease in binding of both ogp160 and sodium metaperiodate-treated ogp160. These data indicate the specificity of the interaction of the deglycosylated form of ogp160 with CXCR4. The results of these experiments suggest that the alteration in the carbohydrate content of the HIV Env molecules resulted in a better exposure of the epitopes involved in gp120 binding to CXCR4. Open in a separate windowFIG. 4Binding of HIV_SF-2 gp120 or the nonglycosylated form, HIV_SF-2 Env 2-3 (Non-glyc SF-2 gp120), to CEM-SS or J25 cells. The concentration was 355 nM for both. The binding of ogp160 and sodium metaperiodate-treated ogp160 (De-glyc ogp160), each at a concentration of 355 nM, to CEM-SS or J25 cells is also shown. The two right-hand bars in each graph show results for cells preexposed to SDF1 at 150 nM. The results are expressed as mean channel fluorescence. The experiments were performed in duplicate with similar results.The understanding of the underlying mechanisms by which HIV Env, CD4, and the newly discovered HIV coreceptors interact to mediate viral entry remains a very significant issue. The way that HIV Env and CD4 interact is well established (28), and some information exists about the interaction between HIV Env, CCR5, and CD4 (34). In this paper we have shown that HIV Env is able to interact in a CD4-independent manner with CXCR4. Still, the extent of such interaction was clearly lower than that of the sCD4-HIV Env complex and CXCR4. This effect of sCD4 seems to be consistent with the observation that the complexing of this molecule with HIV Env from the strains JRFL or BAL resulted in a significant increase in the affinity of HIV Env for CCR5 (34). We speculate that this interaction between sCD4 and HIV Env results in a conformational change that exposes the binding epitopes in HIV Env relevant for binding to CXCR4, as it does with other gp120 epitopes (16). A different scenario would involve a change in both molecules, resulting in a newly formed common binding epitope. This second alternative seems less likely given our data showing CD4-independent binding of HIV Env to CXCR4, as well as previous data showing the existence of HIV strains capable of CD4-independent entry into target cells (9, 15).The gp120 molecule from HIV contains 20 potential N-linked glycosylation sites, with N-linked glycans representing at least 50% of the molecular mass. Their role in CD4 binding has been studied extensively, although some of the results remain somewhat controversial. Most of the available data seem to indicate that complete lack of glycosylation completely (20), or at least partially (25), inhibits HIV Env binding to CD4. Also, enzymatic manipulation of the carbohydrate residues results in a significant decrease but not in complete abrogation of the binding of HIV Env to CD4 (11, 20, 25). It was therefore somewhat unexpected to find that the nonglycosylated form, as well as the sodium metaperiodate-treated form, of HIV Env was able to bind in such an enhanced way to CXCR4. This would appear to reinforce the concept of the existence of a binding epitope for CXCR4 within HIV Env which is different from the one for CD4. It also suggests that the changes occurring as a consequence of the manipulation of the carbohydrate residues likely result in a better exposure of the CXCR4 binding epitope(s) within the HIV Env molecule.In summary, we have shown that HIV Env can interact with CXCR4 in a CD4-independent manner. We have also shown how the interaction of CD4 with HIV Env results in a significant increase in the binding of the latter to CXCR4 and how the alterations in the carbohydrate composition of the HIV Env molecule affect its binding to CXCR4. The complete definition of these interactions may result in novel approaches to protect against cell infection by HIV.     

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M_r of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products. Received: 27 March 1997 / Accepted: 20 May 1997     

Solution structure of the recombinant human oncoprotein p13 MTCP1

The human oncoprotein p13 ^MTCP1 is coded by the MTCP1 gene, a gene involved in chromosomal translocations associated with T-cell prolymphocytic leukemia, a rare form of human leukemia with a mature T-cell phenotype. The primary sequence of p13 ^MTCP1 is highly and only homologous to that of p14 ^TCL1 , a product coded by the gene TCL1 which is also involved in T-cell prolymphocytic leukemia. These two proteins probably represent the first members of a new family of oncogenic proteins. We present the three-dimensional solution structure of the recombinant p13 ^MTCP1 determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After proton resonance assignments, a total of 1253 distance restraints and 64 dihedral restraints were collected. The solution structure of p13 ^MTCP1 is presented as a set of 20 DYANA structures. The rmsd values with respect to the mean structure for the backbone and all heavy atoms for the conformer family are 1.07 ± 0.19 and 1.71 ± 0.17 Å, when the structured core of the protein (residues 11–103) is considered. The solution structure of p13 ^MTCP1 consists of an orthogonal -barrel, composed of eight antiparallel -strands which present an original arrangement. The two -pleated loops which emerge from this barrel might constitute the interaction surface with a potential molecular partner.     

Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modulation of cell cycle progression and of antibody production in mouse hybridomas by a nucleotide analogue

The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20–27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration. This revised version was published online in August 2006 with corrections to the Cover Date.     

New insights into the kinetic resistance to anticancer agents

Kinetic resistance plays a major role in the failure of chemotherapy towards many solid tumors. Kinetic resistance to cytotoxic drugs can be reproduced in vitro by growing the cells as multicellular spheroids (Multicellular Resistance) or as hyperconfluent cultures (Confluence-Dependent Resistance). Recent findings on the cell cycle regulation have permitted a better understanding why cancer cells which arrest in long quiescent phases are poorly sensitive to cell-cycle specific anticancer drugs. Two cyclin-dependent kinase inhibitors (CDKI) seem particularly involved in the cell cycle arrest at the G1 to S transition checkpoint: the p53-dependent p21^cip1 protein which is activated by DNA damage and the p27^kip1 which is a mediator of the contact inhibition signal. Cell quiescence could alter drug-induced apoptosis which is partly dependent on an active progression in the cell cycle and which is facilitated by overexpression of oncogenes such as c-Myc or cyclins. Investigations are yet necessary to determine the influence of the cell cycle on the balance between antagonizing (bcl-2, bcl-X_L...) or stimulating (Bax, Bcl-X_S, Fas...) factors in chemotherapy-induced apoptosis. Quiescent cells could also be protected from toxic agents by an enhanced expression of stress proteins, such as HSP27 which is induced by confluence. New strategies are required to circumvent kinetic resistance of solid tumors: adequate choice of anticancer agents whose activity is not altered by quiescence (radiation, cisplatin), recruitment from G1 to S/G2 phases by cell pretreatment with alkylating drugs or attenuation of CDKI activity by specific inhibitors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Altered expression of flowering class B and class C genes in the Appendix tobacco mutant

 In the tobacco (Nicotiana tabacum) Appendix mutant, anthers are tipped by a miniature style and stigma. The outgrowth appears on the anther when it is already differentiating and follows the developmental timing of the central carpel. The Appendix mutation thus represents a late homeotic transformation suggesting that the APPENDIX (APX) gene either could be a misregulated organ identity gene or could be involved in regulating the expression of such genes. RFLP analysis with two class B (TM6 and NTGLO) and a class C (NAG) probes revealed that the Appendix phenotype is not caused by a mutation in one of these genes. However, in situ hybridization showed important changes in the expression of NTGLO and NAG in the mutant when compared with wild-type tobacco. Surprisingly, although no phenotypic alteration other than the style and stigma outgrowth is observed in the Appendix mutant, changes in class B and class C gene expession were not restricted to the anther tip cells from which the outgrowth originates. As expected, NAG was expressed in the Appendix outgrowth but it was also overexpressed in the normal third and fourth whorl organs at the time the outgrowth, as well as the central styles and stigmas, differentiated. Overexpression of a class C gene is probably responsible for the Appendix phenotype. In normal and mutant flowers, NTGLO was expressed in the second, third and fourth whorls up to the time of carpel fusion. Expression of this class B gene then ceased in the fourth whorl organs but was reactivated at later stages only in the styles and stigmas as well as in the outgrowths of the mutant. It thus seems that the function of the APX gene is either to regulate the late expression of organ identity genes or to control cell proliferation in such a way that, in the mutant, some cells are in a state where they respond in an unusual way to developmental signals. Received: 17 October 1997 / Revision accepted: 24 March 1998