Biomedical subjects
Publications and source records attributed to J Shin.
Three new 9,10-secosteroids, calicoferols C-E [2-4] have been isolated from an undescribed gorgonian of the genus Muricella, and their structures determined by a combination of spectroscopic methods. Calicoferol D [3] exhibited potent antiviral activity and brine-shrimp lethality.
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CD4 and CD8 are members of the immunoglobulin supergene family of proteins, and function as co-receptors with the T cell receptor (TCR) in binding MHC class II or class I molecules, respectively. Within this multimeric complex, CD4 interacts with three distinct ligands. CD4 interacts through its D1 and D2 domains with MHC class II proteins, through its D3 and D4 domains with T cell receptors, and through its cytoplasmic tail with p56lck, a src-related, protein tyrosine kinase. Each of these interactions is important in the function of CD4 and will be discussed in turn.
Ser-42 and Ser-59 in the N-terminal region have been identified as the major phorbol ester-induced phosphorylation sites of p56lck. Phosphorylation of Ser-59 results in a gel shift from 56 kDa to 61 kDa. Simultaneous phosphorylation of Ser-42 and Ser-59 results in a further gel shift to 63 kDa. In vitro kinase assays show that Ser-59 can be uniquely phosphorylated by mitogen-activated protein kinase and that Ser-42 can be phosphorylated by either protein kinase A or protein kinase C.
After synthesis, the alpha chain of the T cell antigen receptor (TCR alpha) can form a complex with other TCR chains and move to the cell surface, or TCR alpha can undergo degradation in the endoplasmic reticulum (ER) if it remains unassembled. The mechanism of translocation and degradation in the ER is unclear. It was found that the putative transmembrane region of TCR alpha (alpha tm) was incompetent on its own to act as a transmembrane region. Molecules that contained alpha tm were translocated into the ER lumen and then underwent either rapid degradation or secretion, depending on the sequence of the cytoplasmic domain. A specific signal for ER degradation within alpha tm does not appear to be present.
We have investigated the role of Ca2+ in the excitation of Limulus photoreceptors by intracellular injection of the Ca2+ buffer, 5,5'-dibromo-BAPTA. Buffer with free Ca2+ of 0.5 or 5 microM slowed the rising edge of the light response over 100-fold and greatly reduced both the transient and plateau phases of the light response, as expected if Ca2+ elevation is necessary for all phases of excitation. Injection of buffers with free Ca2+ of 5 or 45 microM, levels normally reached during light, evoked sustained inward current as expected if Ca2+ is sufficient for excitation. The transduction cascade appears due to a single pathway that sequentially involves 1,4,5-trisphosphate inositol, Ca2+, and cyclic GMP.
CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56lck, a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4-p56lck complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56lck slows the rate of phorbol 12-myristate 13-acetate-induced internalization of CD4 in a manner that depends on a physical association between p56lck and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56lck complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56lck complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.
3,7,11-Trimethyl-13-oxabicyclo[8.3.0]trideca-2,6-diene-12-spiro-2' -(5',5'-dimethyltetrahydrofuran), asperketal B, C20H30O2, M(r) = 302.46, orthorhombic, P2(1)2(1)2(1), a = 10.064(1), b = 22.214(2), c = 8.330 (1) A, V = 1862.3 A3, Z = 4, Dx = 1.08 g cm-3, Cu K alpha, lambda = 1.54178 A, mu = 4.55 cm-1, F(000) = 664, T = 294 (1) K, R = 0.031, wR = 0.027 for 953 data. The backbone of the molecule is composed of a cyclodecadiene ring which is cis fused to a bicyclic ketal system. The relative configurations at C(1), C(10), C(11) and C(12) are 1S*, 10R*, 11R* and 12R*.
By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.
From 1977 through 1985, 113 patients received radiation therapy in conjunction with definitive surgery for adenocarcinoma of the rectum. Posttreatment consisted of a minimum follow-up of 4 years. Radiation was given as postoperative (eight patients), short-course preoperative (2,000 cGy/5 fx, 21 patients), or as full-course preoperative treatment (4,500-5,000 cGy, 84 patients). Three patients received chemotherapy as part of the adjuvant treatment. The local control for the total group was 90% (local failures, 11 of 113), and the rate of recurrence at any site (distant or local) was 30% (34 of 113). Local failure was not significantly influenced by pretreatment clinical findings, tumor grade, or surgical stage. Because of distant failures, overall recurrence was significantly associated with surgical stage--0% (0 of 15) for Astler-Coller A, 23% (7 of 30) for B1, 25% (7 of 28) for B2, and 50% (20 of 40) for B3 or C lesions (p less than 0.01). Locally advanced pretreatment clinical findings were not independent of surgical stage as predictors of outcome. In particular, 14 of the tumors that received full course preoperative radiation were initially either nearly obstructing, circumferential, or deeply fixed. However, by the time of surgery, they were A or B1 lesions (probably down-staged lesions). Only one of 14 (7%) ultimately failed with a local and distant recurrence. There were four cases (3.5%) of small bowel obstruction requiring surgical management. Overall, there were 12 complications (11%) requiring either surgical or major medical management. The complication rate was not associated with radiotherapeutic factors. A strong association was noted between complications and the surgeon. Of 66 patients who had surgery with two colorectal specialists, four (6%) had serious complications. Of the remaining 47 patients who had general surgeons, eight (17%) experienced serious complications.
The cis-acting DNA sequences that control expression of the HLA-DPA and HLA-DQB promoters have been investigated in detail. A set of recombinant plasmids containing 5' promoter deletions as well as site-directed mutants of the DPA and DQB genes were fused to a reporter gene and transfected into human B and interferon gamma (IFN-gamma)-inducible cells. A previously unrecognized cis element, which is essential both for transcription induced by IFN-gamma in several cell types and for constitutive class II expression in B cells, in addition to the well known X and Y boxes, has been defined. This sequence, which spans nucleotides -107 to -98 and -146 to -137 of the DPA and DQB promoters, respectively, has been called the J element. Some evidence for function of a fourth element, the S element, is also presented.
Several structural features of the cytoplasmic domain of CD4 including phosphorylation of Ser-408 have been shown to be important in its endocytosis (Shin, J., Doyle, C., Yang, Z., Kappes, D., and Strominger, J.L. (1990) EMBO J. 9, 425-434). A series of cytoplasmic domain truncations have now indicated that the membrane proximal region of the cytoplasmic domain from Arg-396 to Lys-417 is sufficient for phorbol ester-induced internalization; this segment is predicted to be an alpha-helix. The severe impairment of endocytosis resulting from the mutation Ser-408 to Ala-408 is largely restored by a compensating mutation Ala-404 to Ser-404; phosphorylation of Ser-404 has been directly demonstrated. Furthermore, mutation of Met-407, Ile-410, Leu-413, or Leu-414 to a hydrophilic residue eliminated CD4 endocytosis as did domain truncation at Arg-412. Ser-408 was normally phosphorylated in all of these mutants, suggesting that other residues in this region, including the four hydrophobic amino acids, are also required for CD4 endocytosis. Immunofluorescence microscopy following staining of intact and permeabilized cells showed that all endocytosis defective mutants indeed remained on the cell surface even after phorbol ester treatment, while wild type CD4 was endocytosed and degraded in lysosomes. These data indicate that endocytosis requiring residues 397-417 and binding of lymphocyte tyrosine kinase at residues 417-429 are functions of independent segments of the cytoplasmic region and lead to a hypothesis regarding some features of the endocytic process.
A mutant of CD4 (CD4.Q421stop), in which the cytoplasmic C-terminal 13 amino acids were truncated, was not expressed on the surface of HeLa cells after transfection but was retained in the endoplasmic reticulum (ER). Seven other truncation mutants of CD4 were expressed well on the cell surface, thus suggesting that the C-terminal amino acids of CD4.Q421stop (-Ser-Glu-Lys-Lys-Thr-Cys) may have the sequence information for ER retention. Further mutational study has revealed that two consecutive lysine residues at the third and fourth positions from the C-terminal end are sufficient for ER retention. Lysine at the fourth position, but not at the third position, from the C terminus can be replaced by arginine without disturbing ER retention. Furthermore, two lysine residues at the third and fifth positions from the C terminus also resulted in ER retention. Thus lysine at the third position and a positively charged amino acid either at the fourth or fifth position from the C terminus are sufficient for ER retention of this CD4 mutant, and possibly all transmembrane proteins. In addition to the requirement of specific amino acids at specific positions, the ER retention signal -Lys-Lys-Xaa-Xaa also requires a transmembrane region for function. By contrast -Lys-Asp-Glu-Leu, which targets soluble proteins to the lumen of the ER, does not function in the presence of a transmembrane region.
CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.
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In rats injected with the toxin monocrotaline, altered synthesis and distribution of pulmonary artery elastin suggest that increased elastase activity may be important in the development of vascular changes and progressive pulmonary hypertension. To test this hypothesis, male Sprague-Dawley rats (250-300 g) were given 40 mg/kg of the elastase inhibitor SC-39026 in a carboxymethylcellulose vehicle or vehicle only by gavage, 12 hours before and twice daily for 8 days after a single subcutaneous injection of either monocrotaline (60 mg/kg) or saline. Thirteen days after injection, indwelling cardiovascular catheters were inserted under pentobarbital anesthesia, and at 15 days after injection, pulmonary and systemic hemodynamic measurements were recorded with the animals awake. At post-mortem examination, the lungs were perfused and morphometric techniques applied for light and electron microscopic evaluation. Saline-injected rats given either SC-39026 or vehicle were similar in all features assessed. In contrast, monocrotaline-injected rats given SC-39026 had significantly lower mean pulmonary artery pressure than those given vehicle (21.0 +/- 1.6 vs. 27.5 +/- 0.8 mm Hg, p less than 0.05), and this correlated with a significant reduction in the number of abnormally muscularized arteries at alveolar wall level (r2 = 0.89, p less than 0.001). SC-39026 did not significantly reduce monocrotaline-induced medial hypertrophy of muscular arteries, endothelial injury, and associated subendothelial edema; nor was there a significant increase in the proportion of the medial elastin, although a trend was apparent. Additional groups of monocrotaline injected rats were followed 3 weeks after injection, but both SC-39026 and vehicle-treated rats were similar at this point. Our data suggest that increased serine elastase activity associated with endothelial injury may mediate early abnormal pulmonary vascular smooth muscle differentiation resulting in muscularization of normally nonmuscular peripheral arteries and pulmonary hypertension induced in rats by injection of the toxin monocrotaline. Lack of persistence of this protective effect suggests that there may be continued elastase activity in this model. Failure to inhibit medial hypertrophy with SC-39026 suggests that a different mechanism or a different elastase may be involved in this structural change.