cAMP-dependent protein kinase activation lowers hepatocyte cAMP

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.     

delta- and beta-Crystallin mRNA levels in the embryonic and posthatched chicken lens: temporal and spatial changes during development

The levels of delta- and beta-crystallin mRNAs were examined by cDNA hybridization in the embryonic and posthatched chicken eye lens. Four different cloned beta-crystallin cDNAs were used, allowing discrimination among different members of the beta-crystallin family. Each crystallin mRNA displayed a characteristic temporal and spatial pattern in the developing lens. delta-Crystallin mRNA accumulated rapidly during early embryonic development; by contrast, the beta-crystallin mRNAs began to accumulate rapidly near the end of embryogenesis. Both delta- and beta-crystallin mRNAs increased in the lens for the first month after hatching and began to decrease 3 months after hatching. The levels of the delta- and the different beta-crystallin mRNAs were also differentially regulated in cultured embryonic lens epithelia. The most fiber cell specific crystallin gene product in the differentiating lens was the beta 35 mRNA. These experiments provide a quantitative basis for exploring the differential expression of the delta- and beta-crystallin gene families in the chicken lens.     

The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase.

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.     

Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.     

Secondary plasmodesmata formation in the minor-vein phloem of Cucumis melo L. and Cucurbita pepo L.

Numerous branched plasmodesmata (pd) are present between bundle-sheath cells (BSCs) and specialized companion cells known as intermediary cells (ICs) in the minor-vein phloem of melon (Cucumis melo L.) and squash (Cucurbita pepo L.). These pd were found to be secondary, i.e., they form across existing walls. Sink, sink-source transition, and source tissues were sampled from developing and mature leaves. In sink tissue, IC precursors divide to produce the two to four ICs and associated sieve elements which are present by the time of the sink-source transition. Plasmodesmata along the interface between the IC precursor and adjacent BSCs in sink tissue are unbranched and few in number. Before the leaf tissue undergoes the sink-source transition, the number of pd channels (individual branches of pd) becomes more numerous. This increase in number of pd channels occurs at least in part and perhaps entirely by branching, resulting in more channels on the IC-side than on the BSC-side. In melon there is a 12-fold increase in the number of pd channels within the IC-side of the interface and a corresponding 9-fold increase in pd channels within the BSC-side. Thus, secondary pd form by the time of the sink-source transition and may be involved in phloem loading and photoassimilate export. The system described is well-defined and amenable to experimental manipulation: secondary pd form in large numbers, at a particular interface, over a short period of time, and in a highly predictable manner.Abbreviations BSC bundle-sheath cell - DAP days after planting - IC intermediary cell - LPI leaf plastochron index - pd plasmodesmata - PI plastochron interval We thank Edith Haritatos, Rich Medville, Esther Gowan, and Nancy Dussault for expert technical assistance. This research was supported by an NSF/DOE/USDA Cornell Plant Science Center fellowship (G.M.V.), Natural Sciences and Engineering Research Council Grant GP0138401 and Université de Montréal, Fonds internes de recherche (D.U.B.), and NSF grant IBN-9419703 (R.T.).     

Evolutionary potential of the extrinsic incubation period of dengue virus in Aedes aegypti

Dengue fever is the most common arboviral disease worldwide. It is caused by dengue viruses (DENV) and the mosquito Aedes aegypti is its primary vector. One of the most powerful determinants of a mosquito's ability to transmit DENV is the length of the extrinsic incubation period (EIP), the time it takes for a virus to be transmitted by a mosquito after consuming an infected blood meal. Here, we repeatedly measured DENV load in the saliva of individual mosquitoes over their lifetime and used this in combination with a breeding design to determine the extent to which EIP might respond to the evolutionary forces of drift and selection. We demonstrated that genetic variation among mosquitoes contributes significantly to transmission potential and length of EIP. We reveal that shorter EIP is genetically correlated with reduced mosquito lifespan, highlighting negative life‐history consequences for virus‐infected mosquitoes. This work highlights the capacity for local genetic variation in mosquito populations to evolve and to dramatically affect the nature of human outbreaks. It also provides the impetus for isolating mosquito genes that determine EIP. More broadly, our dual experimental approach offers new opportunities for studying the evolutionary potential of transmission traits in other vector/pathogen systems.     

Evidence that cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation causes pig ovarian granulosa cell differentiation, including increases in two type II subclasses of this kinase

Agents that elevated intracellular cyclic adenosine 3',5'-monophosphate (cAMP) caused a 3- to 10-fold increase in the luteinizing hormone (LH) receptor level and in progesterone biosynthesis in primary cultures of pig ovarian granulosa cells. Associated with these effects was a 2- to 4-fold increase in the total activity of the catalytic subunit of cAMP-dependent protein kinase in the tissue. From quantitation by [3H]cAMP binding and changes in the specific labeling with the photoaffinity analog [32P]-8-azido-cAMP, these agents were found to cause a concomitant 5- to 15-fold increase in two isoforms of the type II R-subunit (Mr = 54,000 and 56,000) of the protein kinase. Since the two intrasubunit cAMP binding sites of the protein kinase have been found to be positively cooperative, the addition of a combination of an analog selective for site 1 and an analog selective for site 2 causes synergistic increases in protein kinase activation in vitro and synergistic increases in intact cell responses if mediated by the cAMP-dependent protein kinase. In the present study, the addition of such a combination of site 1- and site 2-selective analogs to granulosa cells caused a synergistic increase in LH receptor induction and progesterone production. For both responses, synergism did not occur when two analogs selective for the same site were combined. The results indicated that these responses are mediated by either of the two major isozyme types of cAMP-dependent protein kinase.     

c-myc mRNA is elevated as differentiating lens cells withdraw from the cell cycle

Explants of the central region of lens epithelia from early chicken embryos differentiate in vitro to form lens fiber cells when cultured in the presence of chicken vitreous humor. Hybridization of a 32P-labeled v-myc viral oncogene DNA probe to RNA extracted from differentiating explants and immobilized on nitrocellulose filters indicates that levels of 2.5 kb c-myc mRNA are transiently elevated 5-10-fold in the differentiating cells. Increased levels of c-myc mRNA are observed within 30 min of the initiation of differentiation in vitro and persist for 8-9 h. Thymidine labeling of nuclei in differentiating explants indicates that entry of cells into S phase is inhibited during this period, as differentiating cells complete a final round of mitosis and withdraw from the cell cycle. Levels of c-myc mRNA are also elevated in the peripheral region of the lens epithelium, which contains cells undergoing differentiation in vivo, suggesting that the regulation of c-myc mRNA which occurs in vitro may also occur in vivo. c-myc mRNA, c-fos mRNA, and c-src mRNA showed distinct patterns of regulation associated with lens fiber formation in vivo, thus providing evidence that the regulation of c-myc mRNA is specific to this proto-oncogene. The finding that c-myc mRNA undergoes a specific, transient elevation in differentiating lens cells as they withdraw from the cell cycle contrasts with a large body of evidence linking enhanced c-myc expression with increased cell proliferation.     

Transport and steady-state concentration of plasma proteins in the vitreous humor of the chicken embryo: implications for the mechanism of eye growth during early development

The nature and origin of the proteins of the vitreous humor were examined in chickens during embryonic and early posthatching stages. The major proteins of the vitreous humor were similar in electrophoretic mobility to plasma proteins at all ages examined. Earlier studies from our laboratory and experiments described below showed that plasma proteins continuously entered and left the eye throughout its development. From these data it was concluded that the majority of vitreous-humor proteins were derived from the blood. The protein concentration of the vitreous humor was 13% of that of the plasma from embryonic Days 6 through 15 (E6 through E15). After E15, the relative protein concentration in the vitreous humor declined with respect to the plasma and reached 4% of the plasma protein concentration at hatching. Several possibilities were considered to account for how proteins can rapidly enter and leave the eye, yet maintain a steady-state concentration in the vitreous humor that is less than one-seventh of that in the blood.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.