Sequence analysis of leukocyte chemoattractant peptides secreted by periovulatory ovine follicles.

Cells of the white blood series infiltrate ovarian follicles during the ovulatory process. A segment of the wall of periovulatory ovine follicles was incubated and conditioned media subjected to ultrafiltration. Leukocyte chemoattractant activity of media was measured using a linear under-agarose migration assay. Bioactivity was recovered following filtration through a 3000 molecular weight cut-off membrane. Filtrate was then fractionated by reverse phase high performance liquid chromatography. Peptides recovered from two fractions with significant chemoattractant activity were sequenced. One fraction contained 16 amino acid residues with repeating triplets of Gly-X-Y, where X and Y were often proline and hydroxyproline, respectively. Because this motif is characteristic of alpha collagens, and since thecal collagen is degraded during the mechanics of ovulation, it appears that this chemoattractant is derived from the connective tissue matrix of the follicle. Peptide isolated from the other bioactive chromatographic fraction was 15 amino acids in length, and rich in glycine, but did not contain imino acids. To our knowledge this is the first report of purification of leukocyte chemoattractants of reproductive tissue origin. Resident follicular granulocytes and mononuclear cells are capable of secreting a broad spectrum of potent chemicals that could be involved in the mechanisms of ovulation and luteinization.     

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts derived from these patients are extremely sensitive to the mutagenic effect of UV radiation and are abnormally slow in replicating DNA containing UV-induced photoproducts. However, unlike cells from the majority of XP patients, XP variant cells have a normal or nearly normal rate of nucleotide excision repair of such damage. To determine whether their UV hypermutability reflected a slower rate of excision of photoproducts specifically during early S phase when the target gene for mutations, i.e., the hypoxanthine (guanine) phosphoribosyltransferase gene (HPRT), is replicated, we synchronized diploid populations of normal and XP variant fibroblasts, irradiated them in early S phase, and compared the rate of loss of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidones from DNA during S phase. There was no difference. Both removed 94% of the 6-4 pyrimidine-pyrimidones within 8 h and 40% of the dimers within 11 h. There was also no difference between the two cell lines in the rate of repair during G1 phase. To determine whether the hypermutability resulted from abnormal error-prone replication of DNA containing photoproducts, we determined the spectra of mutations induced in the coding region of the HPRT gene of XP variant cells irradiated in early S and G1 phases and compared with those found in normal cells. The majority of the mutations in both types of cells were base substitutions, but the two types of cells differed significantly from each other in the kinds of substitutions, but the two types differed significantly from each other in the kinds of substitutions observed either in mutants from S phase (P < 0.01) or from G1 phase (P = 0.03). In the variant cells, the substitutions were mainly transversions (58% in S, 73% in G1). In the normal cells irradiated in S, the majority of the substitutions were G.C --> A.T, and most involved CC photoproducts in the transcribed strand. In the variant cells irradiated in S, substitutions involving cytosine in the transcribed strand were G.C --> T.A transversions exclusively. G.C --> A.T transitions made up a much smaller fraction of the substitutions than in normal cells (P < 0.02), and all of them involved photoproducts located in the nontranscribed strand. The data strongly suggest that XP variant cells are much less likely than normal cells to incorporate either dAMP or dGMP opposite the pyrimidines involved in photoproducts. This would account for their significantly higher frequency of mutants and might explain their abnormal delay in replicating a UV-damaged template.     

Characterization and partial purification of <Emphasis Type="Italic">Candida albicans</Emphasis> Secretory IL-12 Inhibitory Factor

Background  

We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed.     

Differential cardiac histopathology in inbred mouse strains chronically infected with Trypanosoma cruzi.

Seven inbred mouse strains were examined for the presence of chronic Chagas' cardiomyopathy in postacute Trypanosoma cruzi infection. DBA/1, DBA/2, BALB/c, B10.T (6R), B10.Q, B10.D2, and B6 mice were infected for 100 days with the Brazil strain of T. cruzi. Standard histologic examination of cardiac tissue from these mice revealed the following relationship among the different strains based on the severity of observed inflammation (myocarditis): BALB/c, DBA/1, and DBA/2 were the most inflamed; B10.T (6R) and B10.Q were intermediate; and B6 and B10.D2 showed the least inflammation. Examination of these tissues for characteristics of myocardiopathy such as cell swelling, edema, vacuolization, necrosis, myocytolysis, connective tissue infiltration, and thinning of the right ventricular wall indicated a relative relationship among the different strains relative to the severity of cardiomyopathy as follows: BALB/c, DBA/2, and DBA/1 showed the most cardiopathy (pathopermissive); B10.T (6R) and B10.Q showed intermediate pathology; and B6 and B10.D2 showed the least involvement (pathoresistant). Anti-heart antibody present in the sera of all these mice showed specific reactivity in western blots to a 43-kDa glycoprotein from normal heart tissue. Also, anti-heart antibody enzyme-linked immunosorbent assay titers for all mouse strains were similar and showed no correlation with the severity of tissue damage. The fact that different inbred strains show various degrees of myocarditis and cardiomyopathy may be useful in the study of pathogenesis of chronic Chagas' disease. Results from this limited list of inbred strains suggest that background genes, rather than the major histocompatibility complex, play the major role in the expression of cardiac pathogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.