Molecular cloning and analysis of CDC28 and cyclin homologues from the human fungal pathogen Candida albicans

In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with GI cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional GI cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

Granulocyte colony-stimulating factor modulation of cytokine receptors on murine bone marrow cells. In vivo and in vitro studies.

Human recombinant granulocyte CSF (G-CSF) modulation of cytokine receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo, G-CSF reduced 125I-G-CSF binding to BMC by greater than 95% within 30 min, with return to base line after 48 h. Human rCSF-1 binding was reduced greater than 85% after 30 min and failed to recover even after 48 h. Murine rTNF-alpha or recombinant granulocyte/macrophage CSF binding was not significantly altered. However, human rIL-1 alpha binding increased greater than 1.5-fold after 3 h, was elevated greater than 5-fold between 6 and 12 h, and declined to base line after 48 h. In vitro, G-CSF induced a greater than 1.5-fold increase in IL-1 binding to BMC after 8 h, suggesting that up-modulation of IL-1 binding in vivo required G-CSF and other influences. Further studies indicated that BMC responded to glucocorticoids and G-CSF with a synergistic increase of IL-1 binding. This synergistic IL-1R modulation was a time- and dose-dependent process and was inhibited by cycloheximide or actinomycin D in a dose-dependent manner. Binding studies further revealed that the synergistic stimulation of IL-1R expression on BMC was probably due to increased receptor number, rather than increased receptor affinity. In addition, this phenomenon was also observed in other hematopoietic cells. Our results demonstrated that G-CSF was capable of stimulating IL-1R expression on BMC both in vivo and in vitro and G-CSF in combination with glucocorticoids synergistically up-modulated IL-1 binding to BMC in vitro. Inasmuch as IL-1 induces the secretion of G-CSF and glucocorticoids in vivo, this synergistic induction may play an important, as yet unknown, role in the inflammatory cascade.     

IL-1 modulation of cytokine receptors on bone marrow cells. In vitro and in vivo studies.

We here investigated IL-1 modulation of cytokine receptors on murine bone marrow cells (BMC). In vivo, IL-1 treatment reduced greater than 88% of granulocyte-CSF, greater than 35% of TNF, and greater than 51% of granulocyte/macrophage-CSF binding to BMC after 3 h, and returned to base line after 48 h. However, IL-1 binding to BMC decreased greater than 30% after 30 min, dramatically increased greater than 9-fold between 6 and 10 h, and declined to base line after 48 h. In vitro incubation of BMC with IL-1 did not markedly alter IL-1 and granulocyte-CSF binding, suggesting that modulation of granulocyte-CSF and IL-1 binding to BMC by IL-1 in vivo is due to an indirect mechanism. Further in vitro studies showed that IL-1 binding to BMC was specifically induced by glucocorticoids rather than other steroids, and is a time- and dose-dependent process. IL-1 induced IL-1R up-regulation was suppressed by ketoconazole, cycloheximide, and actinomycin D in a dose-dependent manner. In addition, in vivo dexamethasone imitated the action of IL-1 in stimulating IL-1 binding to BMC and in inducing neutrophilia. Furthermore, IL-1 binding to BMC from sham mice 8 h after IL-1 administration was 2.5 times higher than that observed in adrenalectomized mice. Our results demonstrate, for the first time, that in vivo IL-1-induced increased IL-1 binding to BMC was due to an indirect mechanism, and glucocorticoids stimulated IL-1 binding to BMC in vivo and in vitro. Inasmuch as serum glucocorticoid levels can be elevated by IL-1 in vivo, these results reveal a novel mechanism by which IL-1 modulates its own receptors in vivo.     

Modulation of granulocyte colony-stimulating factor receptors on murine peritoneal exudate macrophages by tumor necrosis factor-alpha.

Modulation of granulocyte CSF (G-CSF) receptors on murine peritoneal exudate macrophages (PEM) by various cytokines was investigated. At 4 degrees C, 125I-G-CSF receptor binding on PEM reached a plateau after 6 h and was specifically competed by unlabeled human rG-CSF but not by other cytokines, including human rG-CSF-1, murine recombinant granulocyte-macrophage CSF, murine rIFN-gamma, human rIL-1 beta, and murine rTNF-alpha. 125I-G-CSF bound to PEM has a half-life of 30 min at 37 degrees C. Preincubation of PEM with murine rTNF, murine recombinant granulocyte-macrophage CSF, CSF-1, or G-CSF for 30 min at 37 degrees C resulted in partial reduction of 125I-G-CSF binding capacity, whereas IL-1 or IFN-gamma did not inhibit G-CSF binding. Further studies indicated that reduction of G-CSF binding caused by TNF was a dose- and time-dependent process and did not require FCS. The reduction was transient, and receptor binding was recovered by incubation at 37 degrees C for 8 h. The recovery of G-CSF binding was inhibited in the presence of cycloheximide. In addition, G-CSF binding studies suggested that the TNF-induced decrease in G-CSF binding to PEM was probably due to a reduction in receptor number rather than receptor affinity. Modulation of G-CSFR by TNF was also observed on nonelicited macrophages from various strains of mice. Our results demonstrate a physiologic response of G-CSFR on macrophages that is modulated by TNF. This phenomenon may play an important, as yet unknown, role in the macrophage inflammatory response.     

Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.     

Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein.

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.     

Carbon Partitioning among Leaves, Fruits, and Seeds during Development of Phaseolus vulgaris L

Development of vegetative and floral buds was found to be a key factor in establishing the way carbon is distributed among growing leaves and fruits in Phaseolus vulgaris L. plants. Leaves emerged principally during a period 14 to 32 days after planting while flowers were produced during a 10- to 12-day period near the end of leaf emergence. Timing of anthesis established the sigmoidal time course for dry weight accumulated by the composite of all fruits on the plant. During the first 12 days following anthesis, fruit growth mainly consisted of elongation and dry weight accumulation by the pod wall. Thereafter, seed dry weight increased for about 1 week, decreased markedly for several days, and then increased again over the next 2 weeks. Accumulation of imported carbon in individual seeds, measured by steady-state labeling, confirmed the time course for dry weight accumulation observed during seed development. Seed respiration rate initially increased rapidly along with dry weight and then remained nearly steady until seed maturation. A number of developmental events described in the literature coincided with the different phases of diauxic growth. The results demonstrated the feasibility of relating current rates of carbon import in individual seeds measured with tracer ¹⁴C to the rates of conversion of imported sucrose and use of the products for specific developmental processes. The resulting data are useful for evaluating the roles of conversion and utilization of imported sucrose in regulating import by developing seeds.