Synthetic peptides including acidic clusters as substrates and inhibitors of rat liver casein kinase TS (type-2)

The hexapeptides AcSer-Glu-Glu-Glu-Val-Glu and Ser-Glu-Glu-Glu-Glu-Glu, reminiscent of the sites phosphorylated by type-2 casein kinase TS in troponin T and glycogen synthase, respectively, have been synthesized and tested as phosphorylatable substrates for casein kinase TS as well as for other protein kinases. Both peptides are readily phosphorylated by casein kinase TS but not, to any detectable extent, by either cAMP-dependent protein kinase or phosphorylase kinase. Phosphorylation by type-1 casein kinase S was almost negligible. On the other hand the hexapeptide Ser-Glu-Glu-Glu-Ala-Ala is phosphorylated much more slowly and the hexapeptide Ser-Glu-Glu-Ala-Ala-Ala is almost unaffected by casein kinase TS. While the Vmax values of casein kinase TS with the acidic hexapeptides are comparable to those obtained with the corresponding protein substrates, the apparent Km values for the peptides are about two orders of magnitude higher than those for the protein substrates. The heptapeptide Arg-Ser-Glu-Glu-Glu-Val-Glu is a very poor substrate of casein kinase TS in comparison with the corresponding hexapeptide lacking the N-terminal Arg; it is, however, a competitive inhibitor toward the protein substrates, exhibiting a Ki similar to those of Ser-Glu-Glu-Glu-Glu-Glu and (Glu)5 which, in turn, are one order of magnitude higher than that of (Glu)10. It is concluded that the minimum structural requirement of type-2 casein kinases consists of a phosphorylatable residue followed by an acidic cluster, whose length is critical for the binding to the enzyme. Additional residues on the N-terminal side are not required, but their nature can influence the transphosphorylation reaction considerably.     

T-cell dysfunction and hyperimmunoglobulinemia E in paracoccidioidomycosis

Various aspects of T and B cell mediated immunity were investigated in 20 well documented cases of active (10) or inactive (10) paracoccidioidomycosis (Pcm), as well as in 8 healthy individuals living in the endemic area of the disease. The results confirm previous reports that active Pcm produces diverse grades of depression of T cell mediated immunity. Such T cell dysfunction is not associated with a reduction in the number of peripheral E rosette-forming cells, and the immunodepression is reversed by chemotherapy. Sera from Pcm (active or inactive) patients have significantly increased levels of total IgE, but the actual proportion of IgE antibodies against P. brasiliensis was very low (0.4–0.6%). The highest levels of total IgE were found in active patients with disease-related immune depression, suggesting that T cell dysfunction might contribute to the excessive IgE production.     

Defective production of anti-herpes simplex virus antibody by neonatal mice. Reconstitution with Ia+ macrophages and T helper lymphocytes from nonimmune adult syngeneic mice

Both neonatal humans and mice are exquisitely susceptible to severe HSV infection. We have now documented a profound defect in the ability of neonatal C57BL/6 mice to produce anti-HSV ADCC antibody. This ability is acquired over the first 2 to 4 wk of life. Reconstitution of neonatal mice by i.p. injection of peritoneal cells from adult nonimmune syngeneic mice both affords dose-dependent protection against lethal HSV infection and reconstitutes the antibody-production defect. By cell-separation techniques (adherence, nylon wool column purification, B cell panning) and cell ablation techniques (silica treatment, irradiation, anti-T cell, anti-Ia, anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies plus complement treatment) the subpopulations involved in the antibody production reconstitution of neonatal mice by adult cells were identified. These include both an Ia+, radioresistant, adherent, silica-sensitive macrophage population and a nylon wool column-purified, radiosensitive, anti-T, anti-Lyt-1.2-sensitive helper T cell population. The latter cell may be substituted for by concanavalin A-stimulated lymphokine-containing spleen cell supernatants or human recombinant IL 2. In addition to reconstitution of ADCC antibody production, the same cell populations, or cells plus lymphokine-containing supernatants or IL 2, protected the newborn mice from lethal HSV infection. Further characterization of this system and of soluble replacement factors has implications for therapy or immunoprophylaxis of human neonates with, or at risk of, HSV infection.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: evidence for mutant forms of folylpolyglutamate synthetase

A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine (CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 (one spontaneous and one Pt(S0₄)₂ induced) have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation (37 °C, pH 7.4 or 9.0) than the parent CHO enzyme. Increased sensitivity of revertant FPGS is observed irrespective of whether one assays the specific catalysis of radioactive tetrahydropteroyldi- or tetraglutamate synthesis. ATP and MgCl₂ protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. AUXB3 FPGS shows a normal sensitivity to 37 °C heat inactivation, but it has an altered substrate saturation and specificity pattern when assayed for tetrahydropteroyldi[U-¹⁴C]glutamate synthesis. Also, unlike the FPGS from parent CHO and a genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra[U-¹⁴C]glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions.     

Involvement of protease in L-glutamine control of nucleic acid and polyphosphate metabolism in cells transformed by 9,10-dimethyl-1,2-benzanthracene, SV40 and H-ras oncogene

Mammalian cells transformed with either 9,10-dimethyl-1,2-benzanthracene, SV40 or H-ras oncogene dramatically changed their ability to synthesize DNA and RNA and metabolize polyphosphate when L-glutamine was withdrawn from the growth medium or when heat shocked (growth at 42 degrees C). Untransformed, DNA and RNA synthesis decreased by 50-80% when glutamine was withdrawn, but polyphosphate accumulated whether or not glutamine was supplied. Heat shock did not alter this response. Transformed isogenic cells responded differently; at 37 degrees C, they decreased their synthesis of DNA and RNA if starved for glutamine, whereas at 42 degrees C, synthesis was optimal without glutamine. Transformed cells accumulated polyphosphate at 37 degrees C when starved for glutamine, but at 42 degrees C, no polyphosphate accumulated. This apparent non-dependence on glutamine by transformed cells when heat shocked was found to be due to the production of glutamine from serum proteins through induction of a protease(s).     

Fine needle aspiration cytology of neoplasms metastatic to the breast

The fine needle aspiration (FNA) cytologic findings in 18 cases of metastatic neoplasms of the breast are reported. The cases were encountered in a combined series of 2,529 FNA breast biopsies, of which 666 were malignant; the metastatic neoplasms of the breast thus constituted 2.7% of all the malignant breast tumors. The series consists of 15 women and 3 men, with a mean age of 48 years (range of 11 to 73 years). Sixteen biopsies confirmed metastatic malignancy in patients with known extramammary primaries; the prebiopsy clinical diagnoses in six of the patients were benign breast lesions. In eight patients, the clinical differential diagnosis was either a benign or malignant primary breast lesion versus a metastatic malignancy. In two additional patients, the FNA biopsy identified metastatic neoplasms from unsuspected extramammary primaries. The metastatic neoplasms included three small-cell carcinomas of the lung, one squamous-cell carcinoma of the lung, two malignant melanomas, three ovarian malignancies, including a dysgerminoma, and one each of carcinoma of the fallopian tube, endometrial carcinoma, transitional-cell carcinoma of the urinary bladder, prostatic carcinoma, acute granulocytic leukemia, lymphoma, mycosis fungoides, hepatoma and neuroblastoma of the retroperitoneum. Recognition of unusual cytologic patterns raised the suspicion of, or confirmed the diagnosis of, malignancy in all cases, with no false-negative diagnoses. None of the cases were cytologically interpreted as a primary breast malignancy. Ancillary studies performed on the FNA material, including immunocytochemistry, contributed to a definitive diagnosis in three cases. FNA diagnosis of metastatic malignancy of the breast is essential in order to avoid unnecessary mastectomy and to ensure appropriate chemotherapy and/or irradiation treatment.     

Preliminary X-ray study of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida N.C.I.B. 9869

Single crystals of p-cresol methylhydroxylase, a flavocytochrome c from Pseudomonas putida, have been prepared. The crystals are orthorhombic, space group P212121 with unit cell parameters; a = 140.3 A, b = 130.6 A and c = 74.1 A. They contain a single non-symmetric dimer per asymmetric unit and diffract to at least 2.5 A resolution.