γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Partial mispairing and crossing-over between β0 and δ genes as the origin of the δ β0 thalassemia gene

Summary Two double heterozygous ⁰/⁰ thalassemic sibs of Mexican descent were studied. The father had a ⁰/⁰ genotype, while the mother, one sib and several maternal relatives were ⁰/⁰ heterozygotes. Parental consanguinity and an apparently low frequency of thalassemia among Mexicans suggested a possible common origin of both ⁰ and ⁰ genes. A hypothesis to explain such a possibility is proposed on the basis of a partial mispairing between ⁰ and genes followed by a crossing-over which would results in a ⁰ recombinant gene. This hypothesis could also be extended to explain the 22 gluala, 22 alaglu and 116 arghis Hb variants as recombinants from double crossing-over between and mispaired genes for which the name interstitial-Lepore is proposed.     

T-cell receptor gene rearrangements and expression in normal human large granular lymphocytes (LGL) and their pathological expansions

The lineage to which normal large granular lymphocytes/natural killer (LGL/NK) cells belong is controversial; in fact they share some surface markers and functional activities with monocytes, but also with T lymphocytes. The relationship of LGL to the T cell lineage by analysis with the T cell receptor (T-rec) gene has been investigated. Pure preparations of human LGL and their CD11⁺ CD8^- and CD11^- CD8⁺ subsets had the T gene in its unrearranged germline configuration. Expression of T and T genes was not detectable. The organization of T gene, which is of particular importance because it occurs early in T cell ontogeny, was also found in its germline configuration.A rare type of lymphoproliferative disorder, termed T-LPD, is characterized by expansion of cells very similar to LGL for morphology, phenotype, and functional activity. Of 17 patients with T-LPD studied for T-rec rearrangement, 15 displayed rearrangement of T and T loci and were CD3+ (14/15 had monoclonal rearrangement), while 2 cases were in germline configuration and were CD3–. Similarly to very small subsets of CD3+ LGL recently described, most T-LPD cases are CD3+ and have T-rec genes rearranged. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage; they also demonstrate that, in contrast to previous views, most TLPD are monoclonal, presumably neoplastic, lymphoproliferative disorders.Abbreviations LGL large granular lymphocytes - NK natural killer - T-rec T-cell receptor - TLPD T lymphoproliferative disease     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat

Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [³⁵S]GTPS with a relatively high affinity (K_d10nM). The binding was saturable and specific as it was indicated by the displacement of bound [³⁵S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [³⁵S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [³⁵S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [³⁵S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - G protein hetero-trimeric GTP binding protein with , , subunits - G_n protein GTP binding protein detected on nitrocellulose blots - GTPS guanosine 5-[-thio]triphosphate - IAA 3-indoleacetic acid - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

The levels of ζ, γ, and δ chains in patients with Hb H disease

Summary Details are given of a study of blood samples from 24 patients with Hb H disease from different Mediterranean countries and from the Far East. Four different types of -thal-1 (--) were observed, namely-() ( 20.5-kb deletion);--MED-I ( 17.5-kb deletion);--MED-II (>26.5-kb deletion); and--SEA ( 18-kb deletion, in Orientals only). The -thal-2 was mainly of the deletion type (16 with the 3.7-kb deletion; 1 with the 4.2-kb deletion), while 4 of the 7 patients with a nondeletional type had the five-nucleotide deletion at the donor splice site of the first intron of the 2 gene. All patients had a mild-to-moderate hemolytic anemia; no significant differences in hematology were observed between the groups. Hb A₂ was decreased to about one-third of the normal level. The Hb H formation varied considerably and its quantitation was not always satisfactory. Patients with Hb H disease due to any -thal-1 combined with a nondeletional -thal-2 had the highest Hb H levels and a more marked anemia. The chain production was small and absent in patients with the MED-II type of -thal-1 because this deletion included the and genes. The highest chain levels were present in the four patients with the SEA type of -thal-1. The chain production was increased, particularly in patients with a mutation of C T at position-158 to the ^G globin gene. This chain was primarily present as Hb Bart's (or ₄) and only about 15% was recovered as Hb F or ₂₂. The evaluation of the rate of chains produced in these patients was greatly facilitated by data from one patient who had Hb H disease and a heterozygosity for the ^A-⁺. The low levels of Hb A₂ and of Hb F (relative to Hb Bart's) can be explained by a decreased affinity of chains for and chains as compared with chains in conditions of severe chain deficiency.     

A new base substitution in the 5′ regulatory region of the humanAγ globin gene is linked with the βs gene

A new TA base substitution, identified inside the 5 regulatory region of the human^A globin gene (^A –499 T A), is reported. This nucleotide change was found to be linked incis with the mutation producing sickle cell anemia (CD6 GAGGTG: ^s gene).     

Secretion of recombinant human fibrinogen by the murine mammary gland

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A, B and fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 g/ml, with total secretion of subunits approaching 700 g/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B and chains were rate limiting. Both the B and chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for chains over B chains. Also, the subunit complexes ₂, A₂ and the individual subunits A, B and were found as secretion products. When the B was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of B chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.     

Interleukin 2-activated killer cells: generation in collaboration with interferonγ and its suppression in cancer patients

Summary The generation of lymphokine activated killer (LAK) cells by recombinant IL2 (rIL2) in collaboration with interferon (IFN) was examined in peripheral blood mononuclear cells (PBMC) from patients with malignant tumors of the digestive organs and breast cancer. LAK cytotoxicity could be induced by rIL2 at 10 units/ml in 10 of 12 patients and 20 of 37 using fresh autologous tumor cells and PK-1, an established solid tumor cell line as a target, respectively. Among 34 patients, in which titers of IFN produced were assayed, 12 showed no IFN production. All of these 12 patients had no or extremely low LAK activity, suggesting the correlation of LAK generation with the production of IFN in response to rIL2. LAK induction by rIL2 in PBMC of cancer patients was almost completely inhibited by addition of anti-IFN serum. Depressed LAK generation, which was accompanied by no or low levels of IFN production, was partially restored by addition of exogenous recombinant IFN. These results indicate that LAK induction by rIL2 in cancer patients involves the production of IFN and its interaction with rIL2.The results also suggested the presence of a factor(s) suppressing LAK induction by rIL2 in the serum of cancer patients. Based on these results, the cancer patients could be divided into the following three groups. Group 1, in which the serum suppressor activity was undetectable, had the same level of LAK cytotoxicity in PBMC as healthy controls. Group 2 showed the serum suppressor factor and had the lower level of cytotoxicity in PBMC when cultivated in autologous serum (AS) compared to healthy controls. The depressed LAK induction in AS medium was restored to a normal level in culture with fetal calf serum (FCS) plus rIL2, or by addition of rIFN, or high concentrations of rIL2 in AS medium. The last group (group 3), in which the serum suppressor factor was also found, had the lowest level of cytotoxicity compared to healthy controls. The LAK induction in these patients could not be restored to a normal level by culture in FCS medium, addition of exogenous rIFN or high concentrations of rIL2, suggesting the possibility that the deficit of LAK generation in this group might involve the dysfunction or the lack of IL2 responder cells, in addition to the presence of a serum suppressor factor(s).     

Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-γ

We investigated the influence of the combined use of sizofiran, a-1,3-glucan and a recombinant interferon- (rIFN-) upon biological activities of peritoneal macrophages (M). The number of peritoneal M and the production of cytokines (interleukin-1, interferon- and tumor necrosis factor) was increased by the combined treatment. Fully activated peritoneal M based on the increased number of elongated pseudopods were observed by electromicroscope. Sizofiran seems to assure a sufficient supply of M to kill tumor cells in the peritoneal cavity and co-administered rIFN- seems to directly stimulate the accumulated M in addition to its direct cytotoxicity against tumor cells. This combination therapy may be a step to the prevention of the recurrence of gynecological malignancies including ovarian cancer, after a negative second-look laparotomy.Abbreviations rIFN- recombinant interferon- - IL-1 interleukin-1 - TNF tumor necrosis factor - SLL second look laparotomy     

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue

The lactone isolated from Fusarium termed L659,699 is a potent specific inhibitor of the enzyme 3hydroxi3methylglutaril coenzyme A (HMG-CoA) synthase. In cultures of smooth muscle cells (SMC) isolated from aortic-arch of control (CSMC) and 5% of cholesterol diet (Ch-SMC) treated chicks, the incorporation of (¹⁴C)acetate to lipids (cholesterol, triacylglycerides and cholesterol ester) were greater in ChSMC cultures than in CSMC and the presence of 0.05 M L659,699 for 2 h in the incubation medium decrease the synthesis of cholesterol however the triacylglycerides synthesis increase. The effect of inhibitor is stronger in young cultures (3–4 steps) than in the older ones (11–12 steps). In young CSMC and ChSMC cultures the inhibition of cholesterol and triacylglycerides synthesis by L659,699 was reversal.     

The human genes for the α and γ subunits of the mast cell receptor for immunoglobulin E are located on human chromosome band 1823

The receptor with high affinity for immunoglobulin E (FcERI) is a key molecule in triggering the allergic reaction. It is tetrameric complex of one subunit, one subunit, and two disulfide-linked subunits. This receptor is present exclusively on mast cells and basophils. Molecules identical to the subunit of FcRI also form cell surface complex with other Fc receptors such as mouse FcRIIa in macrophages and most probably with human FcRIII (CD16) in natural killer (NK) cells. Here we show by in situ hybridization that the human genes for the (FCER1A) and subunits (FCER1 G) of FcERI and the gene for FcRIII (FCGR3, CD16) are located on human chromosome band 1823.     

γ-Glutamylamine cyclotransferase

Summary -Glutamylamine cyclotransferase, an enzyme found in a number of animal tissues and cells, catalyzes the conversion of -(L--glutamyl)-L-lysine to free lysine and 5-oxo-L-proline as well as the release of free amines and the formation of 5-oxo-L-proline from a variety of other L--glutamylamines. Among its substrates are both the mono- and di--glutamyl derivatives of putrescine, spermidine and spermine, and a derivative of -(L--glutamyl)-L-lysine in which both the -amino group and the carboxyl group of the lysine moiety are blocked. The enzyme does not act on most -glutamyl--amino acids, nor is it active toward the -lysyl derivatives of L-aspartic acid or D-glutamic acid. Derivatives of -(L--glutamyl)-L-lysine in which the -amino or the -carboxyl function of the glutamyl moiety is blocked also do not serve as substrates. The specificity of -glutamylamine cyclotransferase is in accordance with the proposal that it functions biologically in the latter stages of the catabolism of products of the action of transglutaminases. Some suggestions as to the manner in which -glutamylamine cyclotransferase serves this function are made based on present knowledge of protein degradation.     

Concurrent effect of visible light on γ-particles,chitin synthetase,and encystment capacity in zoospores ofBlastocladiella emersonii

Summary Zoospores derived from ordinary colorless plants ofBlastocladiella emersonii grown under 240 W/cm² of visible light contain an average of ca. 5 -particles and numerous aggregates of cytoplasmic granules which resemble the -matrix, while spores from dark-grown plants contain ca. 12 -particles but none of the granules. Correspondingly, the amount of chitin synthetase associated with -particles is approximately proportional to the number of -particles in the two spore types. The foregoing light effects, known to be accompanied by an increased capacity for encystment, probably take place before sporogenesis. Conversely, zoospores derived from dark-grown plants do not encyst appreciably over an 8 h period unless they are illuminated, but the number of -particles/spore decreases from 12 to ca. 6 whether or not light is present. Arguments are presented for considering the latter disappearance of -particles as an essential ark reaction which precedes a photochemical reaction, both being needed for light-induced encystment.     

Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.     

Utilization and degradation of lindane by soil microorganisms

Of 147 microorganisms isolated from a loamy sand, 71 showed good growth with lindane (-1,2,3,4,5,6-hexachlorocyclohexane) and produced chloride in an aqueous medium. Thirteen soil microorganisms were selected to study the utilization of lindane. Lindane was metabolized by the microbes to -2,3,4,5,6-pentachloro-1-cyclohexene (-PCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), and pentachlorobenzene (PCB). Cells of Pseudomonas sp. No. 62 grown on lindane simultaneously adapted to -PCCH, -TCCH, -TCCH, -TCCH, PCB, 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB). The bacteria degraded each of these chemicals at least partially as indicated by an increased rate of oxygen consumption.Abbreviations Lindane -1,2,3,4,5,6-hexachlorocyclohexane - -PCCH -2,3,4,5,6-pentachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - PCB pentachlorobenzene - 1,2,3,4-TCB 1,2,3,4-tetrachlorobenzene - 1,2,3,5-TCB 1,2,3,5-tetrachlorobenzene - 1,2,4,5-TCB 1,2,4,5-tetrachlorobenzene - 1,2,3-tCB 1,2,3-trichlorobenzene - 1,2,4-tCB 1,2,4-trichlorobenzene - 1,3,5-tCB 1,3,5-trichlorobenzene - 1,2-DCB 1,2-dichlorobenzene - 1,3-DCB 1,3-dichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene Contribution No. 631, Research Institute, Agriculture Canada, University Sub Post Office, London, Ontario N6A 5B7