Resistance performances of transgenic bt rice lines T(2A)-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The transgenic Bacillus thuringiensis (Bt) rice, Oryza sativa L., lines T(2A)-1 and T1c-19 expressing Cry2A* and Cry1C* from 'Minhui 63' (MH63) were evaluated for resistance to newly hatched and third-instar larvae of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), by using detached leaf laboratory bioassays. Both T(2A)-1 and T1c-19 rice showed high C. medinalis resistance; however, the lethal time (LT)50 of larvae fed with T(2A)-1 rice was significantly longer than that of larvae fed with T1c-19 rice, implying T1c-19 rice was more toxic to C. medinalis larvae. Larval mortality after 4 d on nitrogen-free MH63 was 25.5% compared with 2.4% mortality on high nitrogen fertilizer (250 kg N/ha) plants. Larval mortality on high nitrogen T(2A)-1 plants declined by 20% compared with nitrogen-free plants. However, resistance in T1c-19 plants was unaffected by nitrogen fertilizer. C. medinalis moths preferred MH63 at both the seedling and grain milk stages for oviposition but not the T1c-19 and T(2A)-1 Bt rice lines.     

Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells

Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2(T18AS19A), substitution of both Ser19 and Thr18 by Ala; MRLC2(T18AS19D), Ser19 by Asp and Thr18 by Ala; and MRLC2(T18DS19D), both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2(T18DS19D) contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2(T18AS19D). Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2(T18AS19A) showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells.     

Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells

Steroidal regulation of gene expression in follicular cells is not completely defined. Granulosa cells from 5 mm bovine follicles were cultured and treated and steady-state mRNA levels determined for FSHR (follicle-stimulating hormone receptor) and CYP19A1 (aromatase). Cells were treated for 5 days with (0.1-300 ng/ml) 17beta-estradiol (E2), testosterone (T), or 5alpha-dihydrotestosterone (DHT). FSHR mRNA was increased by T and DHT but not E2. In contrast, CYP19A1 mRNA was induced by all doses of E2 but only high doses of T and DHT. Similarly, varying treatment duration (1-5 days) showed that FSHR was increased by T and DHT and CYP19A1 mRNA increased by E2 and T at all times. Synergism between steroid hormones and FSH or forskolin was also evaluated. FSH or E2 did not alter FSHR mRNA and did not enhance DHT stimulation of FSHR mRNA. In contrast, DHT alone had no effect on CYP19A1 mRNA but synergized with FSH plus E2 to increase CYP19A1 mRNA, probably due to induction of FSHR by DHT. Effects of E2 and T on CYP19A1 were blocked by ICI 182,780, indicating mediation by estrogen receptors. However, the specific androgen receptor antagonist bicalutamide did not block E2 or T effects on CYP19A1 but did block T and DHT stimulation of FSHR. Thus, FSHR is specifically regulated through androgen receptor, whereas CYP19A1 is regulated by multiple pathways, including estrogen receptors and cAMP/protein kinase A induced by FSHR activation in granulosa cells. These inter- and intracellular regulatory mechanisms may be critical for normal follicle growth and dominant follicle selection.     

Hydroxylation of 19-norandrogens by porcine Leydig cells.

The metabolism of 19-norandrogens by porcine Leydig cells was investigated. Non-radioactive 19-norandrostenedione (19-Nor A) and [3H]19-nortestosterone (19-Nor T) were used as substrates in incubations with cell preparations from mature male pigs. Steroid products were separated by reversed-phase HPLC and material in selected peaks was rechromatographed before attempts to identify them by GC-MS. Both 11 beta- and 6 beta-hydroxylated derivatives of 19-Nor A were found and a third product (11-oxo-19-Nor A) was tentatively identified. The profile of radioactive metabolites from [3H]19-Nor T also favours the view of a capacity for hydroxylation of 19-norandrogens by porcine Leydig cells. The significance of these findings together with our earlier report of direct 11 beta-hydroxylation of C19 steroids by such cells remains to be examined.     

Actinopolyspora algeriensis sp. nov., a novel halophilic actinomycete isolated from a Saharan soil

A halophilic actinomycete strain designated H19(T), was isolated from a Saharan soil in the Bamendil region (Ouargla province, South Algeria) and was characterized taxonomically by using a polyphasic approach. The morphological and chemotaxonomic characteristics of the strain were consistent with those of members of the genus Actinopolyspora, and 16S rRNA gene sequence analysis confirmed that strain H19(T) was a novel species of the genus Actinopolyspora. DNA-DNA hybridization value between strain H19(T) and the nearest Actinopolyspora species, A. halophila, was clearly below the 70?% threshold. The genotypic and phenotypic data showed that the organism represents a novel species of the genus Actinopolyspora for which the name Actinopolyspora algeriensis sp. nov. is proposed, with the type strain H19(T) (=?DSM 45476(T)?=?CCUG 62415(T)).     

Functional evaluation of serine/threonine residues in the P-Loop of Rhodobacter sphaeroides phosphoribulokinase.

The N-terminal region of phosphoribulokinase (PRK) has been proposed to contain a "P-loop" or "Walker A" motif. In Rhodobacter sphaeroides PRK, four alcohol side chains, contributed by S14, T18, S19, and T20, map within the P loop and represent potential Mg-ATP ligands. Each of these has been individually replaced with an alanine and the impact of these substitutions on enzyme-ATP interactions and overall catalytic efficiency evaluated. Each mutant PRK retains the ability to tightly bind the positive effector, NADH (0.7-0.9 per site), and exhibits allosteric activation, suggesting that the proteins retain a high degree of structural integrity. Similarly, each mutant PRK retains the ability to stoichiometrically (0.7-1.2 per site) bind the alternative substrate trinitrophenyl-ATP. Despite the large size of the PRK oligomer (8 x 32 kDa), (31)P NMR can be used to detect stoichiometrically bound Mg-ATP substrate, which produces markedly broadened peaks in comparison with signals from unbound Mg-ATP. Elimination of alcohol substituents in mutants T18A, S19A, or T20A produces enzymes which retain the ability to form stable PRKMg-ATP complexes. Each mutant complex is characterized by (31)P resonances for alpha- and gamma-phosphoryls of bound Mg-ATP which are narrower than measured for wild-type PRKMg-ATP; signals for the beta-phosphoryl are poorly detectable for mutant PRKMg-ATP complexes. Kinetic characterization indicates that these mutants differ markedly with respect to catalytic activity. T20A exhibits V(m) comparable to wild-type PRK, while V(m) is diminished by 8-fold for T18A and by 40-fold for S14A. In contrast to these modest effects, S19A exhibits decreases in V(m) and V(m)/K(Ru5P) of 500-fold and >15000-fold, respectively. S19A and T18A exhibit only modest (6-7-fold) increases in S(1/2) for ATP but larger (30-45-fold) increases in K(m) for Ru5P. K(I) values for the competitive inhibitor, 6-phosphogluconate, do not significantly change upon mutation of T18 or S19, suggesting that these residues are not crucial to Ru5P binding. A role for the alcohol group of S19, the eighth residue in P-loop motif, as a ligand to the Mg-ATP substrate seems compatible with the characterization data; adjacent alcohols do not efficiently function as surrogates. Such a proposed function for S19 is compatible with its proximity to E131, the acidic residue in a putative Walker B motif and probable second Mg-ATP ligand in PRK's active site.     

Effects of the regulatory light chain phosphorylation of myosin II on mitosis and cytokinesis of mammalian cells

Myosin plays an important role in mitosis, especially during cytokinesis. Although it has been assumed that phosphorylation of regulatory light chain of myosin (RLC) controls motility of mammalian non-muscle cells, the functional significance of RLC phosphorylation remains uninvestigated. To address this problem, we have produced unphosphorylatable RLC (T18A/S19A RLC) and overexpressed it in COS-7 cells and normal rat kidney cells. Overexpression of T18A/S19A RLC but not wild type RLC almost completely abolished concanavalin A-induced receptor cap formation. The results indicate that myosin phosphorylation is critical for concanavalin A-induced gathering of surface receptors. T18A/S19A RLC overexpression resulted in the production of multinucleated cells, suggesting the failure of proper cell division in these cells. Video microscopic observation revealed that cells expressing T18A/S19A RLC showed abnormalities during mitosis in two respects. One is that the cells produced abnormal cleavage furrows, resulting in incomplete cytokinesis, which suggests that myosin phosphorylation is important for the normal recruitment of myosin molecules into the contractile ring structure. The other is that separation of chromosomes from the metaphase plate is disrupted in T18A/S19A RLC expressing cells, thus preventing proper transition from metaphase to anaphase. These results suggest that, in addition to cytokinesis, myosin and myosin phosphorylation play a role in the karyokinetic process.     

Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire

The Agrobacterium sp. beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.     

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.     

Changes in the testis interstitium of Brown Norway rats with aging and effects of luteinizing and thyroid hormones on the aged testes in enhancing the steroidogenic potential

We tested the possibility of using LH and thyroxine (T(4)) to restore the testicular steroidogenic ability in aged Brown Norway rats. Three-, 6-, 12- (n = 8 per group), and 18-mo-old (n = 32; 3M, 6M, 12M, and 18M, respectively) rats were used. The 18M rats were divided into four groups (n = 8 per group) and implanted subdermally with Alzet mini-osmotic pumps containing saline (control), LH (24 microg/day), T(4) (5 microg/day), and LH+T(4) (24+5 microg/day), respectively, for 4 wk (to 19 mo [19M] of age). Testis volume and absolute volumes of many testicular components were unchanged with advancing age and treatments, except for the blood vessels (occasional thickening), lymphatic space (increased), and Leydig cells (decreased with age but increased to the 3M level with LH and to the 12M level with both T(4) and LH+T(4), respectively). The number of Leydig and connective tissue cells per testis was unchanged with aging and treatments. The number of macrophages was significantly higher in treated rats. The average volume of a Leydig cell was significantly decreased in 12M and 19M control rats. However, LH and LH+T(4) restored it to the 3M level, and T(4) restored to the 12M level. The steroidogenic ability of Leydig cells in vitro decreased when aging from the 3M to the 19M level, LH and T(4) enhanced it to the 12M level, and LH+T(4) raised it to the 3M level. Serum LH was unchanged from 3M to 12M rats, significantly reduced in 19M control rats, and raised above the 3M values with both LH and LH+T(4) treatment and above the 19M (control) values with T(4) treatment; the latter values were lower than the 3M level. Serum T(4) and tri-iodothyronine (T(3)) were highest in 3M and 6M rats and declined in 12M and 19M control rats; the latter group had the lowest levels. In all treated groups, T(4) and T(3) levels were significantly above those of 19M control rats but were lower than those of 3M through 12M rats. Serum testosterone was unchanged from 3M to 12M rats but was reduced in 19M control rats. Both LH and T(4) significantly raised these values above the 19M control levels, but they were still lower than the 3M through 12M levels. Additionally, LH+T(4) significantly raised the serum testosterone levels to those of 12M rats, but these values were significantly lower than those of 3M and 6M rats. These findings show that with 24+5-microg dose of LH+T(4) per day for 4 wk, a 100% recovery of the average volume of a Leydig cell and its steroidogenic ability in vitro and a 73% and 300% restoration of serum testosterone levels compared to 3M and 19M control rats, respectively, could be achieved in aged Brown Norway rats. A 100% reversibility (compared to 3M rats) in serum testosterone levels appears to be possible with adjustments in the LH and T(4) doses in the LH+T(4) treatment.     

Phenotypes and Functions of SARS-CoV-2-Reactive T Cells

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is an ongoing pandemic disease. SARS-CoV-2-specific CD4⁺ and CD8⁺ T-cell responses have been detected and characterized not only in COVID-19 patients and convalescents, but also unexposed individuals. Here, we review the phenotypes and functions of SARS-CoV-2-specific T cells in COVID-19 patients and the relationships between SARS-CoV-2-specific T-cell responses and COVID-19 severity. In addition, we describe the phenotypes and functions of SARS-CoV-2-specific memory T cells after recovery from COVID-19 and discuss the presence of SARS-CoV-2-reactive T cells in unexposed individuals and SARS-CoV-2-specific T-cell responses elicited by COVID-19 vaccines. A better understanding of T-cell responses is important for effective control of the current COVID-19 pandemic.     

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells

Background

The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum.

Characterization of Two Simian Virus 40-Specific RNA Molecules from Infected BS-C-1 Cells

Two discrete simian virus 40 (SV40) RNA species sedimenting at 19 and 16S, respectively, that are present in infected BS-C-1 cells were characterized with respect to the base composition and the ribonuclease T1 fingerprints. The base composition of the 19S SV40 RNA was found to be cytidylic acid (C), 23.0; adenylic acid (A), 28.3; guanylic acid (G), 23.9; and uridylic acid (U), 24.8; that of the 16S SV40 RNA was C, 19.3; A, 34.0; G, 22.0; and U, 24.7 mol%. Analysis of the ribonuclease T1 fingerprints indicated a difference in the base sequence of the 19 and 16S SV40 RNA. The presence of long sequences of adenylic acid residues (poly A) in these viral RNAs was confirmed.     

Molecular Characteristics and Clinical Outcomes of EGFR Exon 19 C-Helix Deletion in Non–Small Cell Lung Cancer and Response to EGFR TKIs

Epidermal growth factor receptor (EGFR) exon 19 deletion (E19del) is the most common activating mutation in advanced non–small cell lung cancer (NSCLC) and associates with the sensitivity of EGFR tyrosine kinase inhibitors (TKIs) treatment. However, not all mutant patterns of E19del have been well studied for the limited coverage of regular EGFR mutation testing. Here, we performed a retrospective cohort study of the C-helix E19del in advanced NSCLC patients based on the screening data by the next-generation sequencing (NGS) platform. From May 2012 to December 2019, clinical information and specimen from 7544 consecutive advanced (IIIB/IV) NSCLC patients were collected and screened for EGFR gene mutations by NGS from multicenters in China. The molecular characteristics and responsiveness to first-line EGFR TKIs therapy in NSCLC patients with C-helix E19del were analyzed. The clinical characteristics were also compared between patients with classical E19del and C-helix E19del. Thirty-eight (2.6%) patients with C-helix E19del and 1400 (97.4%) patients with classical E19dels were identified from 1438 patients with E19del. No significant difference in clinical characteristics was observed between the C-helix E19del and classical E19del groups (P > .05), except for histology (P < .001). All 22 patients with C-helix E19del as p.S752_I759del, p.A750_E758del, p.A750_E758delinsP, p.T751_A755delinsNY, p.T751_I759delinsG, p.T751_I759delinsLD, p.T751_I759delinsN, p.T751_L760delinsNL, and p.T751_D761delinsLY reached the best response as partial response rate (72.7%), and the progression-free survival (PFS) was 12.0 months. The PFS after EGFR TKIs in patients with C-helix E19del tended to be longer than patients with classical E19del but has no statistical significance (12.0 months vs 8.5 months, P = .06). The C-helix E19del could be a positive biomarker for predicting response to EGFR TKIs in advanced NSCLC patients. NGS should be the appropriate platform to identify this rare population, especially when patients harbor no actionable driver mutation initially and are reluctant to accept chemotherapy as first-line therapy.     

Role of cell cycle regulator p19ARF in regulating T cell responses

Although it is well established that the processes of cellular proliferation and apoptosis are linked, the role of cell cycle regulators in T cell responses in vivo is not well understood. In recent years, tumor suppressor molecule p19(ARF) has emerged as a key cell cycle regulator important in cellular apoptosis against strong mitogenic stimuli. In this study, we compared the antigen-specific T cell responses between wild type (+/+) and p19(ARF)-deficient (p19-/-) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). p19-/- mice mounted a potent CD8 T cell response and the magnitude of expansion of LCMV-specific CD8 T cells was comparable to that of +/+ mice. Further, the clonal downsizing of the expanded virus-specific CD8 T cells and establishment of long-term T cell memory were minimally affected by p19(ARF) deficiency. Therefore, p19(ARF) function is not essential to regulate T cell responses following an acute viral infection.     

Two new species of the genus Micromonospora: Micromonospora chokoriensis sp. nov. and Micromonospora coxensis sp. nov., isolated from sandy soil

Two actinomycete strains, 2-19(6)(T) and 2-30-b(28)(T), which produced single, non-motile noduler to warty spore surfaces, were isolated from sandy soil in Chokoria, Cox's Bazar, Bangladesh. A polyphasic study was carried out to establish the taxonomic position of these strains. Morphological and chemotaxonomic characteristics of these strains coincided with those of the genus Micromonospora. Phylogenetic analysis using 16S rDNA sequences indicated that these strains should be classified in the genus Micromonospora. The 16S rDNA sequence of strain 2-19(6)(T )showed closest similarity to the type strains of M. mirobrigensis (98.9%) and M. carbonacea (98.8%), and the strain 2-30-b(28)(T) to the type strains of M. purpureochromogenes (99.4%), M. halophytica (99.3%) and M. aurantiaca (99.2%). Furthermore, a combination of DNA-DNA hybridization results and some differential physiological and biochemical properties indicated that these strains were distinguished from the phylogenetically closest relatives. These strains therefore represent two novel species, for which the name Micromonospora chokoriensis sp. nov. and Micromonospora coxensis sp. nov. are proposed. The type strains are 2-19(6)(T) (=JCM 13247(T) =MTCC 8535(T)) and 2-30-b(28)(T) (=JCM 13248(T)=MTCC 8093(T)).     

Activation of K-RAS by co-mutation of codons 19 and 20 is transforming

The K-RAS oncogene is widely mutated in human cancers. Activating mutations in K-RAS give rise to constitutive signalling through the MAPK/ERK and PI3K/AKT pathways promoting increased cell division, reduced apoptosis and transformation. The majority of activating mutations in K-RAS are located in codons 12 and 13. In a human colorectal cancer we identified a novel K-RAS co-mutation that altered codons 19 and 20 resulting in transitions at both codons (L19F/T20A) in the same allele. Using focus forming transformation assays in vitro , we showed that co-mutation of L19F/T20A in K-RAS demonstrated intermediate transforming ability that was greater than that of individual L19F and T20A mutants, but less than that of G12D and G12V K-RAS mutants. This demonstrated the synergistic effects of co-mutation of codons 19 and 20 and illustrated that co-mutation of these codons is functionally significant.     

Functional heterogeneity among CD4+ encephalitogenic T cells in recruitment of CD8+ T cells in experimental autoimmune encephalomyelitis

Inoculation of Lewis rats with live or attenuated (irradiated or paraformaldehyde-fixed) CD4+ encephalitogenic T cells (S1 line) protects the recipients from transferred experimental autoimmune encephalomyelitis (tEAE) induced by S1 cells. A CD8+ T lymphocyte population specifically activated against the EAE-inducing S1 cells can be readily isolated from the lymphoid organs of pretreated animals. We show, in the present study, that encephalitogenic T cell lines derived from Lewis rats differ in their ability to induce resistance against tEAE in vivo and to stimulate CD8+ cell proliferation in vitro. We also demonstrate that the S19 line of encephalitogenic T cells, in combination with myelin basic protein (MBP), can stimulate CD8+ cell proliferation in vitro. The CD8+ cells generated in this way strongly suppress MBP-specific T cell proliferation in vitro. This combined effect of T cells and MBP was also evident in vivo. Neither S19 cells nor MBP alone induced resistance against S19-mediated tEAE, rather coinjection of these cells and MBP was required. Our results suggest that resistance to EAE is mediated by distinct populations of encephalitogenic T cells that activate Ts cells through different mechanisms. In some instances, both autoreactive T cells and their relevant autoantigen(s) may be needed to activate Ts cells in vivo.     

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)