The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

Isozymes of the glycolytic and pentose-phosphate pathways in storage tissues of different oilseeds

Isozymes of hexose-phosphate isomerase (HPI; EC 5.3.1.9), pyruvate kinase (PK; EC 2.7.1.40) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) have been detected in the developing cotyledons of soybean (Glycine max (L.) Merr.), safflower (Carthamnus tinctorius L.) and sunflower (Helianthus annuus L.). In each seed there are two isozymes each of PK and HPI. The isozyme patterns of 6PGDH are more complex: soybean has two forms of the enzyme, safflower three, and sunflower six. In each tissue, at least 25% of the activity of each of the three enzymes is in the plastids. This supports the proposal that the glycolytic and pentose-phosphate pathways are operating in the plastids and that the plastids are the site of long-chain fatty-acid biosynthesis in developing oilseeds.Abbreviations HPI hexose-phosphate isomerase - 6PGDH 6-phosphogluconate dehydrogenase - PK pyruvate kinase     

Are the in Vitro Effects of Disalicylidenepropanediamine Mediated by Salicylaldehyde?

Disalicylidenepropanediamine (DSPD) has been used widely to investigate photosynthesis using in vitro systems. Evidence is presented which shows that DSPD hydrolyzes rapidly in aqueous solution at pH 7.8 to salicylaldehyde and 1,3-diaminopropane. The effects attributed to DSPD could have been caused by salicylaldehyde.     

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.     

GM-CSF augments the immunosuppressive capacity of neonatal spleen cells in vitro

Addition of exogenous granulocyte-macrophage colony stimulating factor (GM-CSF) to cultures of adult murine spleen cells with sheep red blood cells (SRBC) results in an augmented plaque forming cell (PFC) response. The influence of GM-CSF on the ability of neonatal spleen cells to suppress the anti-SRBC plaque forming response of adult spleen cells was tested by adding GM-CSF to cultures of neonatal and adult spleen cells. The suppressive capacity of the neonatal spleen cells was augmented by exogenous GM-CSF. The augmented suppression of the neonatal spleen cells was dependent on a G-10 adherent population since the addition of GM-CSF to cultures containing G-10 passed neonatal spleen cells resulted in an augmented PFC response and not suppression. Neonatal splenic glass adherent cells were also capable of suppressing the response. Neonatal spleen cells or purified neonatal glass adherent spleen cells cultured in the presence of GM-CSF had markedly increased levels of PGE2 in the culture supernatant. Neonatal spleen cells cultured with GM-CSF had increased numbers of morphologically identifiable macrophages after 48 hr of culture. Both irradiation and G-10 passage of the neonatal spleen diminished the numbers of macrophages formed in response to GM-CSF, and both of these manipulations resulted in reversal of suppression in response to GM-CSF. Thus, the augmented suppressive capacity of neonatal spleen cells in response to GM-CSF is probably mediated by its ability to drive monocyte to macrophage differentiation as well as increase the suppressive capacity of the existing neonatal splenic macrophages by increasing their production of PGE2.     

Identification and localization of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform.

Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.