A 69-kDa membrane protein associated with lipopolysaccharide (LPS)-induced signal transduction in the human monocytic cell line THP-1.

Lipopolysaccharide (LPS) triggers a wide range of cellular responses in mammalian cells. Several proteins, including CD14, have been reported to possess LPS binding capacity. However, the signal transduction molecule(s) and pathway(s) through which LPS induces cellular responses have not been identified. A rabbit antiserum (5299), which had been made against the N-terminal 20-amino-acid sequence of the cytokine, soluble immune response suppressor, unexpectedly activated the human monocyte cell line THP-1 as assessed by tumor necrosis factor (TNF alpha) production. Normal rabbit serum or IgG caused no TNF production, while antiserum 5299 induced TNF at a 1:10(4) dilution. The activity of antiserum 5299 was not due to endotoxin contamination, since antiserum 5299 contained less endotoxin than required for activation of THP-1 and the TNF-inducing activity disappeared when the antiserum was boiled. Moreover, the activity was recovered in purified immunoglobulin G fraction from the antiserum (5299 IgG). These results suggest that antiserum 5299 contains an antibody which recognizes a signal-transducing molecule. THP-1 desensitized by incubation with LPS for 24 hr could not respond to LPS or antiserum 5299 while responsiveness to phorbol ester remained, suggesting that the signals induced by the antiserum 5299 and LPS are transduced through at least a partially common pathway. Western blotting with antiserum 5299 showed a 69-kDa protein which rapidly appeared in Triton X-100-insoluble fraction after stimulation with LPS or 5299 IgG, suggesting an association of 69-kDa protein with the cytoskeleton. Furthermore, two mutant cell lines were isolated (T-15 and T-25) from gamma-irradiated THP-1 which lack responsiveness to LPS. In these mutant cell lines, the 69-kDa protein was not observed in Triton X-100-insoluble fraction after stimulation with LPS. It is proposed that this protein may play an important role in LPS-stimulated signal transduction.

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V alpha 1 gene.

Previous studies of the TCR alpha chain gene have located promoter elements 5' to the start of the various V alpha genes. The only fully characterized enhancer for the entire alpha chain gene (V, J and C genes) has been located approximately 3 kb from the 3' end of C alpha. We now report the existence of additional regulatory elements located in the introns of several murine V alpha genes (V alpha 1, V alpha 3 and V alpha B6.2.16). In the case of V alpha 1, this element appears to be a promoter with bidirectional activity that is not T cell specific. Interestingly, upstream of the promoter in the antisense strand, an open reading frame has been found that codes for a small molecular weight protein (approximately 60 amino acids) that contains a proline-rich region and a tyrosine-isoleucine motif that has homology to Ig beta (the B29 gene product). A rabbit antiserum made against this sequence has confirmed its existence by Western blot and immunoprecipitation. Thus this V alpha 1 intronic promoter has the potential not only to induce the formation of a truncated V alpha 1 gene product, but also regulates the expression of a small molecular weight protein that may be involved in lymphocyte antigen receptor signaling. The activity of this promoter is regulated by changes in intracellular calcium. In the presence of ionomycin the promoter is down-regulated in the sense direction and its activity is enhanced in the antisense direction. This result suggests that this promoter can act differentially to produce two very different gene products. The bidirectional V alpha 1 promoter appears to be the first in the Ig superfamily to induce potentially functional proteins in both directions.

[Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus].

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.

Autoregulation of cochlear blood flow. A comparison of cerebral blood flow with muscular blood flow.

The cochlear blood flow of healthy adult guinea pigs was measured with a laser Doppler flowmeter and flow dynamics were analyzed on the basis of autoregulation. Angiotensin II infusion was used to raise blood pressure, while phlebotomy was done to lower blood pressure. The characteristics of autoregulation of cerebral blood flow and muscular blood flow were also investigated. Cochlear blood flow was considered to have some autoregulation but was less than brain blood flow, which showed significant regulation. Muscular blood flow seemed to have no similar regulatory mechanism.

Effects of a depressor on cochlear blood flow and perilymphatic oxygen tension.

To clarify the characteristics of the blood circulation in the cochlea, we investigated the relationship between cochlear blood flow and perilymphatic oxygen tension in guinea pigs with trimetaphan camsilate induced hypotension. Cochlear blood flow was measured by laser Doppler flowmetry, and perilymphatic oxygen tension by a polarographic method. Cochlear blood flow generally paralleled systemic blood pressure, while perilymphatic oxygen tension showed a slower response to the decrease of systemic blood pressure. Although there were individual differences in the changes of systemic blood pressure, cochlear blood flow and perilymphatic oxygen tension, they were found to be dose dependent. Since hypotension induced by trimetaphan camsilate is fairly reproducible in the dose range of this experiment, this drug can be used as a ganglion blocking agent in experiments on cochlear blood flow and perilymphatic oxygen tension during systemic hypotension. The change of perilymphatic oxygen tension with a slower response could be considered to be a factor in the homeostasis in the inner ear fluid.

Effects of prostaglandins on perilymphatic oxygenation. Enhancement of cochlear autoregulation by prostacyclin.

Effects of prostaglandin E1, prostacyclin, a prostacyclin analogue (OP-2507), and an inhibitor of thromboxane A2 synthetase (OKY-046) on the perilymphatic oxygen tension were measured in the feline tympanic perilymph under various respiratory conditions and blood pressure ranges. Factors influencing the capacity of the autoregulative properties of the inner ear vessels to accommodate changes in systemic blood pressure were also examined. Intravenous administration of prostacyclin, OP-2507, and OKY-046 prevented a decrease in perilymphatic oxygen tension when the systemic blood pressure decreased. Even a negative correlation of the differential coefficients between the perilymphatic oxygen tension and the systemic blood pressure was noticed during the administration of prostacyclin. Thus, prostacyclin enhanced the autoregulative property of the inner ear vessels. Furthermore, correlation studies between perilymphatic oxygen tension and cochlear blood flow demonstrated a partial dissociation, indicating the presence of autoregulation in the mean systemic blood pressure range under 80 mmHg.

[Two cases of surgically treated triple cancer].

Two cases of surgically treated triple cancer are reported. Case 1: Three tumors, one in the lung, one in the stomach, and one in the colon of a 74-year-old man were diagnosed preoperatively. First a subtotal gastrectomy and transverse colectomy were performed and, second 27 days later, a lobectomy of the lower left lung. Pathologically, each tumor proved to be a primary sion. Case 2: A 70-year-old woman, treated surgically for left breast cancer 5 years ago, was found to have anemia. Gastric endoscopy showed a cancer in the antrum. A subtotal gastrectomy was performed. Intraoperatively another tumor in the cecum was found and a right hemicolectomy was also performed. Pathologically each tumor was a primary lesion.