Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin

Follistatin is a glycosylated monomeric protein originally isolated from ovarian follicular fluid based on its ability to specifically inhibit pituitary FSH release. To further explore the physiological role of follistatin, we have expressed recombinant human follistatins with 315 (rhFS-315) and 288 (rhFS-288) amino acids in Chinese hamster ovary cells under the control of the simian virus-40 promoter. The two types of FS originated from alternatively spliced mRNAs and rhFS-315 differed from rhFS-288 by having an extra 27-amino acid sequence at the carboxyl-terminal. The yield of the purified rhFS-315 and rhFS-288 after a single step of affinity chromatography on an activin-coupled Affi-Gel column was 3-5 mg/liter conditioned medium. Using the rhFS-315 and rhFS-288 as molecular mass markers, Western blotting with FS carboxyl-terminal-specific antibodies demonstrated that the majority of native FS isolated from porcine ovarian follicular fluid was neither FS-315 nor FS-288, but was composed of 300 amino acids in various forms of glycosylation. This finding is consistent with our earlier results obtained from tryptic peptide fragment analysis of native FS. Only a very small percentage (less than 1%) of native porcine FS was FS-288. In cultures of rat anterior pituitary cells, rhFS-315 (ED50, 115.2 +/- 16.2 pM) is equipotent to native porcine FS (ED50, 86.7 +/- 14.1 pM) on the suppression of FSH release, but, surprisingly, rhFS-288 (ED50, 9.6 +/- 2.2 pM) is 8-10 times more potent than the native protein, similar to the potency of inhibin-A (ED50, 8.6 +/- 0.9 pM). Interestingly, when the in vivo FSH-suppressing activity of rhFS-288 was compared to that of inhibin-A in 1-week ovariectomized adult rats, it was found that rhFS-288 was more potent and longer acting than inhibin-A. Hence, these results indicate that FS-288 is probably one of the most potent natural FSH suppressors.     

Chemotactic factor for tumor cells derived from the C5a fragment of complement component C5.

Previously, we have stablished that the fifth component of complement (C5) serves as an important source of mediators that have locomotory (chemotactic) activity for leukocytes and tumor cells. C5a, a fragment (Mr 11,200) derived from the NH2-terminal portion of the alpha chain of C5, is the major chemotactic peptide for leukocytes. The present studies demonstrate that cleavage of C5a with trypsin generates a derivative peptide that is chemotactic for tumor cells (Walker carcinosarcoma). This fragment has an estimated Mr of 6000 as assessed by gel filtration and does not require the COOH-terminal arginine of C5a, because equivalent amounts of chemotactic activity for tumor cells can be generated from des-Arg-C5a by digestion with trypsin. The C5a-derived chemotactic peptide for tumor cells demonstrates peak activity at approximately 1 pM. These studies emphasize the key role of the C5a region of the C5 molecule in the generation of peptides that affect locomotory responses of cells.     

Kupffer cells from carbon tetrachloride-injured rat livers produce chemotactic factors for fibroblasts and monocytes: the role of tumor necrosis factor-alpha

Conditioned media from cultured Kupffer and mononuclear macrophagic cells obtained 48 hr after CCl4 administration to rats contains chemotactic factors for human skin fibroblasts and human monocytes. The chemotactic mediator for fibroblasts was approximately 17 kD and was more prominent at early stages of culture. It induced a dose-dependent chemotactic response in fibroblasts. Although the conditioned medium from cultured Kupffer cells of normal rats also contained detectable biological activity, it was significantly less than that in conditioned medium from cultured Kupffer cells from CCl4-treated rats. The activity obtained after purification by high-performance liquid chromatography was completely ablated by incubation with tumor necrosis factor-alpha antibody. Transforming growth factor-beta antibody diminished biological activity by 20%. Human recombinant tumor necrosis factor-alpha and transforming growth factor-beta used in the assay as control showed significant chemotactic activity. The chemotactic activity present in whole normal conditioned medium was only present after 24 and 48 hr of culture. Furthermore, this activity was not neutralized by human recombinant tumor necrosis factor-alpha or transforming growth factor-beta antibodies. Incubation of whole 6-hr conditioned medium with human recombinant tumor necrosis factor-alpha and transforming growth factor-beta antibodies demonstrated and confirmed that tumor necrosis factor-alpha plays a major role in inducing the chemotactic response. On acidification of this supernatant, we found a notable increase in the biological response that could be neutralized by transforming growth factor-beta antibody. Thus tumor necrosis factor-alpha and transforming growth factor-beta may sequentially provide important signals for fibroblast and monocyte recruitment in vivo at initial stages of liver injury.     

Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant.

We have utilized a transendothelial lymphocyte chemotaxis assay to identify and purify a lymphocyte chemoattractant in supernatants of mitogen-stimulated peripheral blood mononuclear cells. Amino acid sequence analysis revealed identity with monocyte chemoattractant protein 1 (MCP-1), a chemoattractant previously thought to be specific for monocytes. Recombinant MCP-1 is chemoattractive for purified T lymphocytes and for CD3+ lymphocytes in peripheral blood lymphocyte preparations. The T-cell response to MCP-1 is dose-dependent and chemotactic, rather than chemokinetic. Phenotyping of chemoattracted T lymphocytes shows they are an activated memory subset. The response to MCP-1 by T lymphocytes can be duplicated in the absence of an endothelial monolayer and the majority of T-lymphocyte chemotactic activity in mitogen-stimulated peripheral blood mononuclear cell supernatants can be neutralized by antibody to MCP-1. Thus, MCP-1 is the major lymphocyte chemoattractant secreted by mitogen-stimulated peripheral blood mononuclear cells and is capable of acting as a potent T-lymphocyte, as well as monocyte, chemoattractant. This may help explain why monocytes and T lymphocytes of the memory subset are always found together at sites of antigen-induced inflammation.     

Migration of human leukocytes from soft agarose droplet: A simplified method for studying chemotaxis and spontaneous migration

Studies of in vitro chemotaxis and spontaneous migration of human leukocytes using the accepted method with the Boyden-chamber-filter are troublesome, because of the need for specially constructed vessels as well as the difficulties caused by the use of membrane filters. We describe a new and simplified method for measuring human leucocyte chemotaxis, which is a modification of the recently described underagarose migration method and which is based upon spontaneous migration of cells from a soft agarose droplet and in response to a chemotactic gradient. We examined suspensions of leukocytes, purified granulocytes, and mononuclear cells from 10 healthy normal adults and from 10 samples of cord blood using E Coli O₁₁₁B₄ endotoxin-activated human serum as attractant. Our results showed that the mean chemotactic indices (C.I.-chemotaxis/migration) for purified granulocytes and for mononuclear cells from normal individuals were 3.0 ± 1.2 and 2.7 ± 1.5, respectively. Chemotaxis was significantly reduced when unwashed leukocytes were studied, indicating a detrimental effect of autologous plasma on leukocytic response to a chemotactic stimulus in this system. Cord blood cells showed normal spontaneous migration, but significantly decreased chemotaxis. This preliminary report shows that the technique is simple, rapid, and reproducible, and can detect abnormalities of chemotaxis in both granulocytes and mononuclear cells.     

Functional receptors for insulin-like growth factors I and II in rat thymocytes and mouse thymoma cells

Functional receptors for insulin-like growth factors (IGF) I and II have been identified in rat thymocytes and mouse thymoma cell lines R1.1 and S49.1. IGF-I receptor alpha-subunit (MW 130,000) bind IGF-I and IGF-II with equal affinity (Kd approximately 4-7 nM), and insulin with approximately 100 times lower affinity. Tyrosine kinase activity and autophosphorylation of the IGF-I receptor beta-subunit (MW 95,000) are stimulated by IGF-I and IGF-II with equal potency (ED50 approximately 0.5 nM). IGF-II receptors (MW 250,000) bind IGF-II with Kd approximately 0.3 nM and IGF-I with 30 times lower affinity, but not insulin. IGF-I and IGF-II do not cross-react with the insulin receptor to which insulin binds with an apparent Kd approximately 1 nM, and stimulates its tyrosine kinase activity with ED50 approximately 3 nM. In thymocytes, alpha-aminoisobutyric acid transport is stimulated 2-fold by IGF-I and IGF-II with identical potency (ED50 approximately 2 nM), and by insulin with ED50 approximately 10 nM. Activation of thymocytes by concanavalin A increased the number of IGF-II receptors 2-fold, whereas IGF-I receptor binding and IGF-stimulated amino acid transport were unaltered. We conclude that the effect of IGF-I and IGF-II in thymocytes is mediated via binding to the IGF-I receptor and stimulation of its tyrosine kinase. The presence of functional IGF receptors on thymocytes and thymoma cells suggests that IGF-I and IGF-II play a role in the regulation of thymic functions.     

Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF.

The two major mitogenic polypeptides for endothelial cells have been purified to homogeneity. The complete primary structure of bovine pituitary basic fibroblast growth factor (FGF) and the amino-terminal amino acid sequence of bovine brain acidic FGF have been established by gas-phase sequence analyses. Homogeneous preparations of these polypeptides are potent mitogens (basic FGF, ED50 approximately equal to 60 pg/ml; acidic FGF ED50 approximately equal to 6000 pg/ml) for many diverse cell types including capillary endothelial cells, vascular smooth muscle cells, and adrenocortical and granulosa cells; in vivo, basic FGF is a powerful angiogenic agent in the chick chorioallantoic membrane assay. The available protein sequence data demonstrate the existence of significant structural homology between the two polypeptides.     

Defective mononuclear leukocyte chemotaxis in the Chediak-Higashi syndrome of humans, mink, and cattle

Chemotaxis of mononuclear leukocytes from humans, mink, and cattle was evaluated in vitro using a morphologic Boyden chamber technique and a new 51-Cr-labeled mononuclear radioassay with a double micropore filter system. Significantly decreased mononuclear leukocyte chemotactic response were noted when human, mink, or cattle Chediak-Higashi cells were tested using autologous serum or endotoxin-activated autolotous serum. A similar Chediak-Higashi mononuclear leukocyte defect was noted in humans when kallikrein or dialyzable transfer factor were used as the chemotactic stimulus. Studies using smaller pore filters in the chemotactic chamber exaggerated the chemotactic defect. Serum from Chediak-Higashi subjects had normal chemotactic activity. Additional studies on the spontaneous (random) locomotion of Chediak-Higashi mononuclear leukocytes revealed normal results when a capillary tube assay system was used, but abnormal results were obtained when a Boyden chamber micropore filter assay was used, demonstrating fundamental differences in these two assays of random locomotion. It is clear from these studies that defective mononuclear leukocyte chemotaxis is another feature of the imparied host defenses in the Chediak-Higashi syndrome that may contribute to the marked susceptibility to pyogenic infections so characteristic of this dease.     

Type beta transforming growth factor: a bifunctional regulator of cellular growth.

Type beta transforming growth factor (TGF-beta) is a two-chain polypeptide of 25,000 daltons isolated from many tissues, including bovine kidney, human placenta, and human platelets. It has been characterized by its ability to stimulate reversible transformation of nonneoplastic murine fibroblasts, as measured by the formation of colonies of these cells in soft agar (ED50 = 4 pM TGF-beta for NRK fibroblasts). We now show that the response of cells to TGF-beta is bifunctional, in that TGF-beta inhibits the anchorage-dependent growth of NRK fibroblasts and of human tumor cells by increasing cell cycle time. Moreover, the anchorage-independent growth of many human melanoma, lung carcinoma, and breast carcinoma cell lines is inhibited by TGF-beta at concentrations in the same range as those that stimulate colony formation of NRK fibroblasts (average ED50 = 10-30 pM TGF-beta for inhibition). Whereas epidermal growth factor and TGF-beta synergize to induce anchorage-independent growth of NRK fibroblasts, their effects on the growth of A-549 human lung carcinoma cells are antagonistic. The bifunctional response of cells to TGF-beta is further demonstrated in Fischer rat 3T3 fibroblasts transfected with a cellular myc gene. In these cells TGF-beta synergizes with platelet-derived growth factor to stimulate colony formation but inhibits the colony formation induced by epidermal growth factor. The data indicate that the effects of TGF-beta on cells are not a function of the peptide itself, but rather of the total set of growth factors and their receptors that is operant in the cell at a given time.     

Pulsatile release of gonadotropin-releasing hormone from hypothalamic explants is restrained by blockade of N-methyl-D,L-aspartate receptors

We have shown previously that N-methyl-D,L-aspartate (NMDA) and kainate, two neuroexcitatory amino acids acting through distinct receptors, may induce the release of GnRH from hypothalamic explants. However, that effect could have no physiological significance, since very high concentrations (50 mM) of NMDA and kainate were required. Here, using agents blocking the activation of receptors to neuroexcitatory amino acids, we evaluated their possible physiological involvement in the pulsatile release of GnRH from the hypothalamus of 50-day-old male rats in vitro. In control conditions (10 nM glycine and 1 mM mg2+), the release of GnRH in 7.5-min fractions collected for 2-4 h showed an obvious pulsatile pattern. The mean (+/- 1 SD) interval between pulses, identified by PULSAR program, was 34.3 +/- 11.4 min. The stimulation of GnRH release by NMDA (50 mM) added to the medium for 7.5 min could be blocked reversibly in the presence of MK-801 (100 microM) using medium without glycine or enriched with Mg2+ (2 mM). The endogenous pulses of GnRH secretion were abolished in the presence of MK-801 or using increased Mg2+ concentrations as well as in the absence of glycine. In contrast, pulsatile release of GnRH was not affected in the presence of 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), a selective inhibitor of kainate and quisqualate receptors which suppressed the increase in GnRH release induced by kainate (50 mM) without affecting the response to NMDA. These data indicate that the physiological mechanism of pulsatile GnRH secretion in the hypothalamus may involve endogenous neuroexcitatory factors acting through NMDA-sensitive receptors.     

Inflammatory cells in bronchial asthma

A diagrammatic representation of the interactions between mediators of hypersensitivity and leukocytes in early, late-phase, and ongoing asthma is shown in Figure 1. Early phase or immediate reactions are largely the result of bronchoconstriction consequent to the release of mediators such as histamine, PGD2, LTC4/D4, and PAF. The principal mediator cell (MC) is the mast cell (although other IgE receptor-bearing cells such as the macrophage, eosinophil, and platelet might also be involved in this immediate response). The stimulus for mediator cell activation may be either immunologic (IgE-dependent) or nonimmunologic (i.e., changes in osmolarity as a result of the respiratory water loss associated with exercise-induced asthma). Late-phase reactions appear to be a consequence of infiltration with neutrophils (N), eosinophils (E), and macrophages (M phi). These cells are recruited and activated either by mast cell-associated chemotactic factors [such as LTB4, PAF, the eosinophil chemotactic factor of anaphylaxis (ECF-A), or high-molecular weight neutrophil chemotactic activity (NCA (HMW))] and/or "lymphokines" derived from T-helper cells (TH) which have been stimulated by antigen processed by the antigen-processing cells (APC). These mononuclear cell interactions are under the control of regulatory T cells [T suppressor (TS) cells] and it is speculated that the availability of these subsets may determine the magnitude of the late-phase response. Lymphokines and monokines which selectively activate neutrophils, eosinophils, and monocytes include LIF, EAF, and IFN-gamma, respectively. Macrophage-derived tumor necrosis factor (TNF) also amplifies the inflammatory response by its capacity to enhance eosinophil cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-ricin B chain conjugate has enhanced biological activity in insulin-insensitive cells

Insulin-ricin B chain conjugate, a hybrid molecule consisting of insulin covalently linked to the binding chain of ricin, was tested for insulin-like biological activity in HTC and H35 rat hepatoma cells, rat adipocytes, rat thymocytes, and human fibroblasts. In H35 cells and adipocytes, cells that have abundant insulin receptors and are very sensitive to insulin (ED50 of 30 pM and 50 pM, respectively), the conjugate had 5% the biological activity of native insulin (ED50 of 500 pM and 1000 pM, respectively). Since the insulin portion of the conjugate has 5% the potency of native insulin in binding to the insulin receptor, these observations suggested that (in these cells) the conjugate was acting via the insulin receptor. Moreover lactose and galactose, potent inhibitors of ricin binding to its receptor, had no effect on the action of the conjugate on H35 cells. In contrast, in thymocytes, HTC cells, and fibroblasts cells that have relatively few insulin receptors and require high concentrations of insulin to elicit biological actions (ED50 of 10 nM, 20 nM, and 1 nM, respectively), the conjugate had more biological activity than was predicted on the basis of its ability to bind to the insulin receptor. In addition, in these three insulin-insensitive cell types, the activity of the conjugate was inhibited by either lactose or galactose. Thus, these observations indicate that in cells which are relatively insensitive to insulin, the biological effects of the conjugate require the interaction of the ricin B chain moiety with the ricin receptor. In addition, in fibroblasts, the activity of the conjugate was inhibited by the addition of a monoclonal antibody to the insulin receptor which inhibits the response of fibroblasts to insulin. These data suggest, therefore, that in insulin-insensitive cells the binding of the ricin B chain moiety to its receptor enhances the interaction of the insulin portion of the conjugate with the insulin receptor.     

Identification of specific inhibin A-binding proteins on mouse Leydig (TM3) and sertoli (TM4) cell lines

The binding of human inhibin A to cell surface binding proteins of mouse Leydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysis identified two classes of inhibin A-binding sites on TM3 (K(d(1)) = 85 pM and 4,160 sites/cell; K(d(2)) = 520 pM and 12,500 sites/cell) and TM4 (K(d(1)) = 61 pM and 2,620 sites/cell; K(d(2)) = 520 pM and 10,400 sites/cell) cells. Compared with inhibin A, inhibin B only partially competed [(125)I]inhibin A binding (6-8%), whereas activin A competed weakly (<0.01%). Chemical cross-linking of [(125)I]inhibin A to both cell lines identified five [(125)I]inhibin A binding complexes with apparent molecular masses of 70, 95, 145, 155, and more than 200 kDa. Inhibin A displacement of [(125)I]inhibin A from each of these cross-linked species (ED(50) = 60-110 pM) closely resembled displacement from intact TM3 (ED(50) = 97 +/- 32 pM) and TM4 (ED(50) = 75 +/- 28 pM) cells, suggesting that all of these proteins are involved in the high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human betaglycan identified protein complexes of more than 200, 145, and 95 kDa. It is concluded that the high affinity binding complex for inhibin A found in these cell lines consists of betaglycan and several proteins of unknown identity and may represent the putative inhibin receptor complex.     

Cation-induced restoration of insulin action in insulin-desensitized HTC cells.

Insulin desensitization of amino acid uptake in HTC cells was induced by preincubation with 4 or 10 micrograms/ml insulin. Insulin binding after desensitisation was decreased by both insulin concentrations due to a 45-49% decrease in insulin receptor numbers. Desensitization with 4 micrograms/ml insulin increased the ED50 for half-maximal stimulation of amino acid uptake from 19.5 +/- 9.2 ng/ml in control cells to 84.0 +/- 8.3 ng/ml (P less than 0.0001). It also decreased the maximal insulin response of amino acid uptake from 1.40 +/- 0.10 to 1.14 +/- 0.10 nmol/mg protein, indicating the production of a mild postreceptor defect. Desensitization with 10 micrograms/ml insulin completely abolished this insulin response. When cellular receptors were down-regulated with 4 micrograms/ml insulin and restimulated with insulin in the presence of 0.03 mM ruthenium red (RR) or 10 mM Ca2+, both the insulin response and insulin binding were increased. Insulin binding was restored to levels comparable to those observed in control cells by an increase in receptor affinity. The ED50 of amino acid uptake decreased to 20.5 +/- 7.3 ng/ml insulin in the presence of RR and to 42.2 +/- 8.9 ng/ml in the presence of 10 mM Ca2+ (both P less than 0.0001 from down-regulated cells). In addition, the maximal insulin response increased from 1.14 +/- 0.10 to 1.40 +/- 0.10 and 1.45 +/- 0.10 nmol/mg protein, respectively. Preincubation with 10 micrograms/ml insulin prevented the effect of RR and Ca2+ on the recovery of insulin responses. These experiments suggest that insulin-desensitized cells undergo a progressive loss of their insulin response and that RR and Ca2+ provide useful reagents to investigate the mechanisms of this process because they can counteract the decrease in insulin response by increasing receptor affinity and receptor-effector coupling.     

Androgens exert selective effects on protein kinase C induced LH and FSH release in rat anterior pituitary cells in culture

This study compares the selective effect of androgens on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release induced either by LH-releasing hormone (LH-RH) or by protein kinase C activation in rat anterior pituitary cells in culture. In control cells, phorbol-12-myristate-13-acetate (PMA) stimulated the release of radioimmunoassayable LH and FSH in a dose-dependent manner at an ED50 value of 5.95 +/- 1.45 nM. The maximal release of gonadotropins induced during a 3-hour incubation with PMA was 40-60% of that induced by LH-RH. Preincubation of the cells for 2-4 days with 10 nM 5 alpha-dihydrotestosterone (DHT) decreased by approximately 60% the subsequent release of LH induced by 100 nM LH-RH or by 500 nM PMA during a subsequent 3-hour incubation. The inhibitory effect of DHT was completely suppressed by coincubation with the antiandrogen hydroxyflutamide. DHT exerted a similar inhibitory effect on LH release induced by another stimulator of protein kinase C activity, namely 1-oleoyl-2-acetylglycerol. The potent inhibitory action of DHT on LH-RH- or PMA-induced LH release was exerted at an ED50 value of approximately 10 pM. Contrary to the effect on LH, the FSH response to LH-RH or to PMA was increased by preincubation with 2 nM DHT, the stimulatory effect of the androgen being completely antagonized by hydroxyflutamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic characteristics of luteinizing hormone release from perifused sheep anterior pituitary cells stimulated by combined pulsatile and continuous gonadotropin-releasing hormone

We aim to quantify and relate the dominant dynamic factors of GnRH signals and of the resultant patterns of LH release from pituitary cells. Using perifused sheep cells we have already shown that rising edges of GnRH pulses are major effectors of LH release and that a longer absence of signal between pulses improves response. This study reports the effects on LH release dynamics of continuous levels of GnRH with superimposed pulses and of slowing the important rising edge of the GnRH pulses. Low baseline GnRH perifusions at physiological levels (5-60 pM) reduced the response to hourly pulses of 850 pM GnRH. Continuous GnRH (420 pM), which initially yielded maximal LH release followed by desensitization, prevented extra stimulation by pulses of equal concentration, but 10-fold higher pulses gave additional LH output. After desensitization an hour's respite from stimulation resensitized cells to 420 pM pulses. Whereas continuous stimulation of cells with GnRH even at the very low level of 5-10 pM [ED50 = 58 +/- 6 (SE) pM] produced desensitization in 10-15 min, slowly rising GnRH (0.56-14 pM/min) caused increasing LH output with time. However, in comparison with square wave pulses, stimulatory signals consisting of slowly rising concentrations of GnRH produced peaks characterized by less total LH output and a changed shape. This was consistent with desensitization at low concentrations of GnRH reducing response to later increases in the level of stimulation. The mechanism for detecting GnRH signals and/or the mechanisms controlling release of LH were desensitized to constant GnRH at any concentration but retained a reduced sensitivity, or developed an additional release capacity, to increased levels of GnRH. Properties of four distinct types of LH release dynamics were described quantitatively and were shown to be controlled by different time constants in the GnRH pulse stimulation patterns.     

Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes.

A cDNA clone encoding a human serine esterase gene was isolated from a library constructed from poly(A)+ RNA of allogeneically stimulated, interleukin 2-expanded peripheral blood mononuclear cells. The clone, designated HSE26.1, represents a full-length copy of a 0.9-kilobase mRNA present in human cytotoxic cells but absent from a wide variety of noncytotoxic cell lines. Clone HSE26.1 contains an 892-base-pair sequence, including a single 741-base-pair open reading frame encoding a putative 247-residue polypeptide. The first 20 amino acids of the polypeptide form a leader sequence. The mature protein is predicted to have an unglycosylated Mr of approximately equal to 26,000 and contains a single potential site for N-linked glycosylation. The nucleotide and predicted amino acid sequences of clone HSE26.1 are homologous with all murine and human serine esterases cloned thus far but are most similar to mouse granzyme B (70% nucleotide and 68% amino acid identity). HSE26.1 protein is expressed weakly in unstimulated peripheral blood mononuclear cells but is strongly induced within 6-hr incubation in medium containing phytohemagglutinin. The data suggest that the protein encoded by HSE26.1 plays a role in cell-mediated cytotoxicity.     

Coagulopathy in amyloidosis: Combined deficiency of factors IX and X

Combined severe deficiencies of blood clotting factors IX and X were observed in 2 patients who suffered from systemic amyloidosis. This unique deficiency state was marked by refractoriness to Vitamin K as well as to transfusion therapy. Increased antithrombin activity was present in both individuals and corresponded in time to the emergence of a monoclonal IgG kappa light chain paraprotein in 1. Both patients demonstrated profound bleeding disorders. It is hypothesized that the Vitamin K dependent factors have special affinity for amyloid deposits due to an unusual amino acid (γ-carboxyglutamic acid) present in these factors.     

Oxaloacetate as the hill oxidant in mesophyll cells of plants possessing the c(4)-dicarboxylic Acid cycle of leaf photosynthesis

Isolated mesophyll cells from leaves of plants that use the C(4) dicarboxylic acid pathway of CO(2) fixation have been used to demonstrate that oxaloacetic acid reduction to malic acid is coupled to the photochemical evolution of oxygen through the presumed production of NADPH. The major acid-stable product of light-dependent CO(2) fixation is shown to be malic acid. In the presence of phosphoenolpyruvate and bicarbonate the stoichiometry of CO(2) fixation into acid-stable products to O(2) evolution is shown to be near 1.0. Thus oxaloacetic acid acts directly as the Hill oxidant in mesophyll cell chloroplasts. The experiments are taken as a firm demonstration that the C(4) dicarboxylic acid cycle of photosynthesis is the major pathway for the fixation of CO(2) in mesophyll cells of plants having this pathway.