Physiological comparisons of two soybean cultivars differing in canopy photosynthesis. I. Variation in vertical 14CO2 labelling and dry weight partitioning

Differences in canopy apparent photosynthesis (CAP) among soybean [Glycine max (L.) Merr.] genotypes have been shown to be correlated to seed yields. Since the physiological basis for such differences in CAP is unknown, two cultivars known to differ in CAP, Tracy and Davis, were studied during the 1978–1980 growing seasons. The CAP and dry weights of component plant parts were determined. In 1978 and 1979, ¹⁴CO₂ uptake by vertical leaf strata was determined and specific leaf weight (SLW) and leaf area index (LAI) were determined for corresponding strata in 1979 and 1980. Measurements were taken on several dates during reproductive growth. With the exception of CAP, all measurements (¹⁴C uptake, dry weights) were made in layers within the canopy. CAP on some dates were significantly higher in Tracy than in Davis and integrated CAP values from a certain growth period, labeled as R5 to R7, averaged 16 percent higher in Tracy for the three years studied. No differences in the relative recovery of ¹⁴C from different layers of leaves in the canopy were found. This indicates that variations in canopy structure or leaf orientation did not play a major role in the CAP differences between cultivars. The differences seem related to variations in leaf dry weights. Overall, Tracy exhibited 13.5, 19.2, and 13.2 percent greater leaf dry weights than Davis during 1978, 1979, and 1980, respectively. These differences in leaf dry weight seem largely due to a differences in the SLW. Data from these experiments indicate that differences in soybean CAP values were associated with differences in SLW.Abbreviations CAP Canopy Apparent Photosynthesis - CER Carbondioxide Exchange Rates - EST Eastern Standard Time - LAI Leaf Area Index - LSD Least Significant Difference - POPOP 1,4-bis-[2(5-phenyloxazdyl)]-benzene - PPO 2,5-diphenyloxazole - SLW Specific Leaf Weight     

Species‐specific microsatellite markers to monitor gene flow between exotic poplars and their natural relatives in eastern North America

Species‐specific microsatellite markers were obtained for the unambiguous recognition of five poplar species of ecological and commercial importance to eastern North America: the native species Populus balsamifera and Populus deltoides, the exotic species Populus maximowiczii, Populus nigra, Populus trichocarpa and their interspecific hybrids. Forty‐four of 71 tested primer pairs amplified simple sequence repeat (SSR) loci for all five taxa. Six of these loci showed non‐overlapping allelic diversity between species, including fixed differences. Together, they were useful to identify unambiguously the five taxa and to validate parental contributions in a group of hybrid progeny. These markers will be invaluable to detect gene flow from plantations of exotic poplar into adjacent stands of native species and between the two potentially hybridizing native species P. balsamifera and P. deltoides.     

DNA-Binding activity of bis-netropsin containing a cis-diaminoplatinum group between two netropsin fragments

The binding of Pt-bis-Nt and its modified analog Pt*-bis-Nt, which has two additional glycine residues in the linker between two netropsin fragments, to DNA has been studied. The elongation of the linker in the bis-netropsin molecule increases the cytotoxicity and leads to an almost complete loss of the antiherpetic activity of bis-netropsin. The study of the binding of two bis-netropsins to an oligonucleotide duplex containing an AT cluster, which is present at the origin of replication of herpes virus (OriS), revealed significant structural differences between the complexes of bis-netropsins with this DNA oligomer. It was shown by CD spectroscopy that the binding of Pt-bis-Nt in the extended conformation and in hairpin form with the parallel orientation of two bis-netropsin fragments makes a greater contribution to the interaction with the duplex than in the case of Pt*-bis-Nt. At high binding levels, Pt*-bis-Nt binds to the AT cluster in OriS predominantly in the form of associates based on the antiparallel, double-stranded, pyrrolcarboxyamide motif. The interaction of Pt-bis-Nt and Pt*-bis-Nt with a single-stranded oligonuclotide (64 nt) corresponding to the upper strand at the origin of replication of herpes virus (OriS*) was also studied. Substantial differences in the binding of bis-netropsins to OriS* and the thermostability of the resulting complexes were found by CD spectroscopy and UV melting studies.     

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease.     

Protein dynamics viewed by hydrogen exchange

To examine the relationship between protein structural dynamics and measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nuclease was compared with that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursion that removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motion and the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.     

Antioxidant protective effect of flavonoids on linoleic acid peroxidation induced by copper(II)/ascorbic acid system

Antioxidants are compounds that can delay or inhibit lipid oxidation. The peroxidation of linoleic acid (LA) in the absence and presence of Cu(II) ion–ascorbate combinations was investigated in aerated and incubated emulsions at 37 °C and pH 7. LA peroxidation induced by copper(II)–ascorbic acid system followed first order kinetics with respect to hydroperoxides concentration. The extent of copper-initiated peroxide production in a LA system assayed by ferric thiocyanate method was used to determine possible antioxidant and prooxidant activities of the added flavonoids. The effects of three different flavonoids of similar structure, i.e. quercetin (QR), morin (MR) and catechin (CT), as potential antioxidant protectors were studied in the selected peroxidation system. The inhibitive order of flavonoids in the protection of LA peroxidation was: morin > catechin ≥ quercetin, i.e. agreeing with that of formal reduction potentials versus NHE at pH 7, i.e. 0.60, 0.57 and 0.33 V for MR, CT, and QR, respectively. Morin showed antioxidant effect at all concentrations whereas catechin and quercetin showed both antioxidant and prooxidant effects depending on their concentrations. The structural requirements for antioxidant activity in flavonoids interestingly coincide with those for Cu(II)-induced prooxidant activity, because as the reducing power of a flavonoid increases, Cu(II)–Cu(I) reduction is facilitated that may end up with the production of reactive species. The findings of this study were evaluated in the light of structure–activity relationships of flavonoids, and the results are believed to be useful to better understand the actual conditions where flavonoids may act as prooxidants in the preservation of heterogeneous food samples containing traces of transition metal ions.     

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA⁺ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.     

Grouping in auditory temporal perception and vocal production is mutually adapted: the case of wriggling calls of mice

Auditory Gestalt perception by grouping of species-specific vocalizations to a perceptual stream with a defined meaning is typical for human speech perception but has not been studied in non-human mammals so far. Here we use synthesized models of vocalizations (series of wriggling calls) of mouse pups (Mus domesticus) and show that their mothers perceive the call series as a meaningful Gestalt for the release of instinctive maternal behavior, if the inter-call intervals have durations of 100–400 ms. Shorter or longer inter-call intervals significantly reduce the maternal responsiveness. We also show that series of natural wriggling calls have inter-call intervals mainly in the range of 100–400 ms. Thus, series of natural wriggling calls of pups match the time-domain auditory filters of their mothers in order to be optimally perceived and recognized. A similar time window exists for the production of human speech and the perception of series of sounds by humans. Neural mechanisms for setting the boundaries of the time window are discussed.     

Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-). METHODS: A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone. RESULTS: Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation. CONCLUSIONS: This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.