Soil Carbon Pools in Dryland Agroecosystems as Affected by Several Years of Drought.

No-till and increased cropping intensity (CI) can increase yield and soil organic C (SOC) in the US Great Plains compared with traditional wheat ( L.)-fallow management. However, gains in SOC and other C pools may not be permanent. Increasing frequency of drought may reduce C inputs and potentially reverse gains accrued during wetter periods. This study examined the effect of drought on the persistence of SOC with two objectives: (i) to determine soil C pools (0-20 cm) after 24 yr in no-till as influenced by potential evapotranspiration (PET), landscape position (slope), and CI; and (ii) to compare the size of the C pools after the first 12 yr (wet) versus the subsequent 12 yr, notable for frequent droughts. Rotations were wheat-corn ( L.)-fallow (WCF), continuous cropping (CC), and a grass Conservation Reserve Program mixture planted across slopes at three sites in Colorado with similar precipitation but increasing PET. After 24 yr, water-soluble organic C increased with CI from WCF to CC to grass with 250, 340, and 440 kg C ha, respectively. Soil microbial biomass C also increased with CI-1500, 1660, and 2135 kg C ha for WCF, CC, and grass, respectively. The particulate organic matter C pool had a three-way interaction with PET, slope, and CI. Overall, between Years 12 and 24, SOC increased in grass by 16.9%, with a rate of 425 kg C ha yr sequestration compared with 10.5 and 1.4% for the WCF and CC systems, respectively.

The crystal structure of TGF-beta 3 and comparison to TGF-beta 2: implications for receptor binding.

Transforming growth factors beta belong to a group of cytokines that control cellular proliferation and differentiation. Five isoforms are known that share approximately 75% sequence identity, but exert different biological activities. The structure of TGF-beta 3 was solved by X-ray crystallography and refined to a final R-factor of 17.5% at 2.0 A resolution. Comparison with the structure of TGF-beta 2 (Schlunegger MP, Grütter MG, 1992, Nature 358:430-434; Daopin S, Piez KA, Ogawa Y, Davies DR, 1992, Science 257:369-373) reveals a virtually identical central core. Differences exist in the conformations of the N-terminal alpha-helix and in the beta-sheet loops. In TGF-beta 3, the N-terminal alpha-helix has moved approximately 1 A away from the central core. This movement can be correlated with the mutation of Leu 17 to Val and Ala 47 to Pro in TGF-beta 3. The beta-sheet loops rotate as a rigid body 9 degrees around an axis that runs approximately parallel to the dimer axis. If these differences are recognized by the TGF-beta receptors, they might account for the individual cellular responses. A molecule of the precipitating agent dioxane is bound in a crystal contact, forming a hydrogen bond with Trp 32. This dioxane may occupy a carbohydrate-binding site, because dioxane possesses some structural similarity with a carbohydrate. The dioxane is in contact with two tryptophans, which are often involved in carbohydrate recognition.

Crystallization and preliminary X-ray analysis of recombinant human transforming growth factor beta 2.

Recombinant human transforming growth factor beta 2 (TGF-beta 2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed-phase HPLC. The recombinant TGF-beta 2 was shown to have a biological activity equal to that of native TGF-beta 2 in a fibroblast migration assay. Pure, active recombinant TGF-beta 2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3(1)21 or P3(2)21 have unit cell dimensions of a=b=60.6 A, c=75.2 A and diffract beyond 2.0 A.

Transforming growth factors type-beta and dexamethasone attenuate group II phospholipase A2 gene expression by interleukin-1 and forskolin in rat mesangial cells.

Treatment of rat mesangial cells with interleukin-1 beta (IL-1 beta) and forskolin induced, in a synergistic fashion, the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2. In contrast, interleukin-6 did not increase PLA2 mRNA levels of PLA2 activity. Transforming growth factor (TGF) beta 1, TGF beta 2 and TGF beta 3 equipotently attenuated the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. The glucocorticoid dexamethasone only partially suppressed the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, but totally inhibited PLA2 synthesis and secretion.

Selective cloning of B cell hybridoma-specific rearranged immunoglobulin gene loci using the polymerase chain reaction.

The use of conventional DNA cloning procedures to obtain productively rearranged Ig genes from B cell hybridomas for structure/function analysis of immunoglobulins is tedious and time-consuming. Here we describe a procedure based on PCR which permits rapid, selective isolation of DNA segments containing individual hybridoma-specific Ig gene rearrangements. The method, an adaptation of the so-called 'inverted PCR' technique (IPCR), can be applied most efficiently to specific genes where a preliminary restriction map is available from Southern blot analysis of the hybridoma genomic DNA. To achieve amplification of a given rearranged Ig locus, small amounts of total hybridoma DNA are digested to completion with a chosen restriction endonuclease and the fragments circularised by DNA ligase. Cleavage of the DNA circles using a second restriction enzyme, chosen specifically to cut 3' to a rearranged V-(D)-J exon, leads to linear DNA segments where the rearranged gene is now flanked by segments of known nucleotide sequence derived originally from the 3' region of the Ig H or L chain gene locus. This permits the selection of oligonucleotides that provide convergent primers for specific amplification of DNA segments containing the required gene rearrangement. Amplified DNA fragments can be cloned and rapidly characterised by sequence analysis.

New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion.

The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.

The replicative restriction of lymphocytotropic isolates of HIV-1 in macrophages is overcome by TGF-beta.

In vitro exposure of human blood monocyte-derived macrophages to T-cell tropic human immunodeficiency virus (HIV) isolates fails to establish a productive viral infection. Several studies have shown that such preferential HIV-1 replication in T cells or in mononuclear phagocytes (HIV tropism) may be determined by distinct viral characteristics. In the present study it was demonstrated that transforming growth factor-beta (TGF-beta), a factor known to be produced by platelets, macrophages, and other cells present at a wound site, can act as a mediator in overcoming the lymphocytotropic restriction of several well-characterized viral isolates of HIV-1 (i.e., LAV, Z84, pLAI, NY5). Macrophages infected with these isolates show cytopathic changes comparable to those seen upon infection with the monocytotropic isolate ADA. To achieve this effect with TGF-beta, the factor must be present after the infection period. The emerging virus retains its original cellular tropism. Based on these observations the authors propose a role for TGF-beta in the establishment and progression of HIV infection and disease.

Wound healing in aged animals--effects of locally applied transforming growth factor beta 2 in different model systems.

Local application of a growth factor which could stimulate cell turnover, extracellular matrix synthesis and blood vessel formation in the skin should improve and accelerate wound healing processes which are often impaired in old age. We demonstrate the effects of TGF-beta 2 in promoting wound repair in old animals where normal healing responses are shown to be naturally slower. The potential use of TGF-beta s for the treatment of wound injuries, including chronic non-healing ulcers in the elderly, is discussed.

Localization of transforming growth factor-beta 1, -beta 2 and -beta 3 gene expression in bovine mammary gland.

We have studied the expression of transforming growth factor (TGF)-beta 1, -beta 2, and -beta 3 in the non-lactating and lactating bovine mammary gland by in situ hybridization. All three isoforms were expressed in the lobuloalveolar framework of the non-lactating and lactating gland although marked differences were apparent in their spatial distribution. TGF-beta 1 was expressed predominantly by the epithelial cells of the lobules although expression was also observed in the intralobular stroma cells lining the epithelium. In contrast, TGF-beta 2 expression was only observed in the epithelial cells. TGF-beta 3 transcripts were expressed at the highest levels and were observed in almost all cells of the lobule. No TGF-beta signals were found in the interlobular regions of the mammary gland. The possible regulatory functions of these molecules in development of the mammary gland and on differentiation processes in the neonate are discussed.

In situ hybridization analysis of cytokine, proto-oncogene and tumour suppressor gene expression in psoriasis.

The purpose of this study was to investigate and to compare, by in situ hybridization, gene expression of IL-1 beta, IL-8, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-alpha, p53 and c-myc in lesions and in non-involved skin of patients with psoriasis. All lesional skin biopsies showed overexpression of IL-1 beta, IL-8 TGF-alpha mRNAs. IL-1 beta hybridization signals were strong in a small number of cells localized predominantly in the dermal papillae and in the suprapapillary epidermis. Overexpression of TGF-alpha was observed in all suprabasal keratinocytes, whereas strongly elevated IL-8 mRNA expression was found to be restricted to clusters of suprabasal keratinocytes. TGF-beta 3, p53 and c-myc transcripts were clearly detected in the epidermis of all biopsies, although expression levels were comparable in lesional and non-lesional skin.

ERG oncoprotein expression in prostate cancer: clonal progression of ERG-positive tumor cells and potential for ERG-based stratification.

Gene fusions prevalent in prostate cancer (CaP) lead to the elevated expression of the ERG proto-oncogene. ERG activation present in 50-70% of prostate tumors underscores one of the most common oncogenic alterations in CaP. Despite numerous reports of gene fusions and mRNA expression, ERG oncoprotein status in CaP still remains to be defined. Furthermore, development of ERG protein-based assays may provide a new dimension to evaluation of gene fusions involving diverse androgen-regulated promoters and the ERG protein-coding sequence. Through exhaustive evaluations of 132 whole-mount prostates (261 tumor foci and over 200 000 benign glands) for the ERG oncoprotein nuclear expression, we demonstrated 99.9% specificity for detecting prostate tumor cells using a highly specific anti-ERG monoclonal antibody. The ERG oncoprotein expression correlated well with fusion transcript or gene fusion in randomly selected specimens. Strong concordance of ERG-positive foci of prostatic intraepithelial neoplasia (PIN) with ERG-positive carcinoma (82 out of 85 sections with PIN, 96.5%) affirms the biological role of ERG in clonal selection of prostate tumors in 65% (86 out of 132) of patients. Conversely, ERG negative PINs were associated with ERG-negative carcinoma. Taken together, the homogeneous and strong ERG expression detected in individual tumors establishes the potential for ERG oncoprotein-based stratification of CaP.

Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange.

We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 50 degrees. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double- and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel.

Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A.

Monoclonal antibodies directed against rabbit reticulocyte protein synthesis initiation factor 4A (eIF-4A) were used to isolate mouse cDNA clones expressing eIF-4A protein sequences in E. coli. The identity of cDNA clones encoding eIF-4A sequences was confirmed by hybrid-selected translation and peptide mapping of the translation product. Analysis of the mRNA coding for eIF-4A from mouse liver and HeLa cells by Northern hybridization revealed two discrete mRNA species of approximately 2000 and 1600 nucleotides in length. The existence of two mRNAs in mouse and HeLa cells encoding eIF-4A was confirmed by cDNA sequencing.

DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice.

We have determined the complete nucleotide sequence of the DNA of the immunosuppressive variant of the parvovirus minute virus of mice (MVMi) and compared it to the published sequence (12) of the fibroblast-specific strain (MVMp). We have found 175 differences between the two viruses, most of which affect single nucleotides. Despite these differences, the genomic organization of MVMp and MVMi is identical. There are 29 amino-acid changes between the putative viral gene products of MVMi and MVMp, 16 of which are conservative. We discuss the possibility that the differential tissue-specificity of the two variants is linked to differences within the non-transcribed region near the 5' end of the viral genomes.

A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates.

In analogy to the Escherichia coli replicative DNA polymerase III we define two forms of DNA polymerase alpha: the core enzyme and the holoenzyme. The core enzyme is not able to elongate efficiently primed single-stranded DNA templates, in contrast to the holoenzyme which functions well on in vivo-like template. Using these criteria, we have identified and partially purified DNA polymerase alpha holoenzyme from calf thymus and have compared it to the corresponding homogeneous DNA polymerase alpha (defined as the core enzyme) from the same tissue. The holoenzyme is able to use single-stranded parvoviral DNA and M13 DNA with a single RNA primer as template. The core enzyme, on the other hand, although active on DNAs treated with deoxyribonuclease to create random gaps, is unable to act on these two long, single-stranded DNAs. E. coli DNA polymerase III holoenzyme also copies the two in vivo-like templates, while the core enzyme is virtually inactive. The homologous single-stranded DNA-binding proteins from calf thymus and from E. coli stimulate the respective holoenzymes and inhibit the core enzymes. These results suggest a cooperation between a DNA polymerase holoenzyme and its homologous single-stranded DNA-binding protein. The prokaryotic and the mammalian holoenzyme behave similarly in several chromatographic systems.

Comparison of canine parvovirus with mink enteritis virus by restriction site mapping.

The genomes of canine parvovirus and mink enteritis virus were compared by restriction enzyme analysis of their replicative-form DNAs. Of 79 mapped sites, 68, or 86%, were found to be common for both types of DNA, indicating that canine parvovirus and mink enteritis virus are closely related viruses. Whether they evolved from a common precursor or whether canine parvovirus is derived from mink enteritis virus, however, cannot be deduced from our present data.

A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus.

We have determined the exact splicing patterns of the mRNAs of the minute virus of mice by a combination of cDNA sequencing and S1 nuclease protection analysis. There are four virus-specific mRNA species, each coding for one of the four polypeptides identified by in vitro translation. The R1 mRNA comprises sequences from nucleotide approximately 200 to 2281 and from 2378 to approximately 4800 and codes for the NS1 protein. The R2 mRNA is derived from nucleotides approximately 200 to 515, 1991 to 2281, and 2378 to approximately 4800 and codes for the NS2 protein. Between nucleotides 1991 and 2281, the coding sequence for NS2 overlaps that of NS1, but in a different reading frame. R3 covers nucleotides approximately 2007 to 2281 and 2378 to approximately 4800 and codes for VP2. The fourth species, R3', differs from R3 by using an alternative splice donor and acceptor in the region around 47 map units (nucleotide 2400); it extends from nucleotide approximately 2007 to 2317 and from 2400 to approximately 4800 and almost certainly codes for VP1. The R2 transcript is unusual in that the intron that was removed from it (nucleotides 516 to 1990) starts with GC rather than the canonical GU. With the exception of the splice acceptor at position 2378, which is found only in rodent parvoviruses, the splice junctions are highly conserved among autonomous parvoviruses. These results show that minute virus of mice, like other small DNA viruses, uses multiple strategies to compress the coding information for several viral proteins into a short (5,104 nucleotide) genome.