Summary The pathogenic fungus Cryptococcus neoformans produces melanin within its cell wall for infection and resistance against external stresses such as exposure to UV, temperature fluctuations and reactive oxygen species. It has been reported that melanin may also protect cells from ionizing radiation damage, against which C. neoformans is extremely resistant. This has tagged melanin as a potential radioprotective biomaterial. Here, we report the effect of melanin on the transcriptomic response of C. neoformans to gamma radiation. We did not observe a substantial protective effect of melanin against gamma radiation, and the general gene expression patterns in irradiated cells were independent of the presence of melanin. However, melanization itself dramatically altered the C. neoformans transcriptome, primarily by repressing genes involved in respiration and cell growth. We suggest that, in addition to providing a physical and chemical barrier against external stresses, melanin production alters the transcriptional landscape of C. neoformans with the result of increased resistance to uncertain environmental conditions. This observation demonstrates the importance of the melanization process in understanding the stress response of C. neoformans and for understanding fungal physiology.

The present study tests the hypothesis that chronic hypoxia enhances reactivity to nitric oxide (NO) through age-dependent increases in soluble guanylate cyclase (sGC) and protein kinase G (PKG) activity. In term fetal and adult ovine carotids, chronic hypoxia had no significant effect on mRNA levels for the beta1-subunit of sGC, but depressed sGC abundance by 16% in fetal and 50% in adult arteries, through possible depression of rates of mRNA translation (15% in fetal and 50% in adult) and/or increased protein turnover. Chronic hypoxia also depressed the catalytic activity of sGC, but only in fetal arteries (63%). Total sGC activity was reduced by chronic hypoxia in both fetal (69%) and adult (37%) carotid homogenates, but this effect was not observed in intact arteries when sGC activity was measured by timed accumulation of cGMP. In intact arteries treated with 300 microM 3-isobutyl-1-methylxanthine (IBMX), chronic hypoxia dramatically enhanced sGC activity in fetal (186%) but not adult (89%) arteries. This latter observation suggests that homogenization either removed an sGC activator, released an sGC inhibitor, or altered the phosphorylation state of the enzyme, resulting in reduced activity. In the absence of IBMX, chronic hypoxia had no significant effect on rates of cGMP accumulation. Chronic hypoxia also depressed the ability of the cGMP analog, 8-(p-chlorophenylthio)-cGMP, to promote vasorelaxation in both fetal (8%) and adult (12%) arteries. Together, these results emphasize the fact that intact and homogenized artery studies of sGC activity do not always yield equivalent results. The results further suggest that enhancement of reactivity to NO by chronic hypoxia must occur upstream of PKG and can only be possible if changes in cGMP occurred in functional compartments that afforded either temporal or chemical protection to the actions of phosphodiesterase. The range and age dependence of hypoxic effects observed also suggest that some responses to hypoxia must be compensatory and homeostatic, with reactivity to NO as the primary regulated variable.

The present study tests the hypothesis that age-dependent increases in endothelial vasodilator capacity are due to maturational increases in endothelial nitric oxide (NO) synthesis and release. Intact 4-cm carotid artery segments taken from term fetal lambs and nonpregnant adult sheep were perfused by using a closed system that enabled independent control of flow and inflow pressure and facilitated complete recovery of all NO released. Fluid shear stress induced a graded release of NO (in nmol NO x min x cm(-2) of luminal surface area) that was significantly greater in adult (890 +/- 140) than in fetal (300 +/- 40) carotid arteries at corresponding values of shear stress (5.9 +/- 0.3 dyn/cm2) but was independent of inflow pressure in both age groups. These age-related differences in NO release were not attributable to corresponding differences in endothelial NO synthase (eNOS) abundance, as eNOS protein levels (in ng of eNOS/cm2 of luminal surface area) were similar in adult (14 +/- 2) and fetal (12 +/- 1) arteries. Adult (80 +/- 15) and fetal (89 +/- 32) levels of eNOS mRNA (in 10(6) copies/cm2 of luminal surface area) were also similar. However, when NO release was normalized relative to the associated mass of eNOS protein to estimate eNOS-specific activity in situ, this value (in nmol NO x microg of eNOS(-1) x min(-1)) was significantly greater in adult (177 +/- 44) than in fetal (97 +/- 36) arteries when the endothelium was maximally activated by A-23187. Similarly, the slope of the relation between fluid shear stress and estimated eNOS-specific activity (in nmol NO x microg of eNOS(-1) x min(-1) per dyn/cm2) was also significantly greater in adult (6.8 +/- 0.1) than in fetal (2.9 +/- 0.1) arteries, which suggests that eNOS may be more sensitive to or more efficiently coupled to activating stimuli in adult compared with fetal arteries. We conclude that maturational increases in endothelial vasodilator capacity are attributable to age-dependent increases in NO release secondary to elevated eNOS-specific activity and involve more efficient coupling between endothelial activation and enhancement of eNOS activity in adult compared with fetal arteries.

Acclimatization to chronic hypoxia involves numerous compensatory changes in many tissues, including blood vessels. The present data demonstrate that in addition to well-documented changes in contractility, chronic hypoxia also produces important changes in the mechanisms mediating endothelium-dependent vasodilatation. At the level of the endothelium, hypoxia attenuates endothelial release of NO and this appears to be mediated through reductions in eNOS specific activity; chronic hypoxia has little effect on eNOS abundance. In contrast, chronic hypoxia depresses the abundance of sGC, which functions as the downstream vascular receptor for NO released from the endothelium. The decreased abundance of sGC produced by chronic hypoxia occurs without changes in sGC specific activity and results in decreased rates of NO-induced cGMP synthesis. Nonetheless, the vasodilator efficacy of NO is enhanced in hypoxic arteries, which suggests that mechanisms downstream from sGC are upregulated by hypoxia. Consistent with this view, chronic hypoxia significantly depresses PDE activity, which serves to prolong cGMP half-life and enhance its vasodilator effects. It remains possible that chronic hypoxia may also enhance PKG activity and/or the abundance of its substrates; this possibility remains a promising topic for future investigation. Overall, it is important to recognize that the mechanisms of adaptation to chronic hypoxia identified in the present study may be somewhat unique to adult carotid arteries. Adaptive responses to chronic hypoxia can vary considerably between small and large arteries, and also between immature and adult arteries . Still, the present data clearly demonstrate that both the endothelium and vascular smooth muscle of major arteries are profoundly influenced by chronic hypoxia, and thereby participate fully in whole-body adaptation to reduced oxygen availability.

Growth and differentiation-related pathways are much more active in immature than in mature, fully differentiated smooth muscle. Because mitogen-activated protein kinases (MAPK) are intimately involved with growth and differentiation, and the extracellular signal-regulated kinase (ERK) subfamily of MAPKs are involved in some contractile responses, the present studies examined the hypothesis that ERKs play an important and age-dependent role in smooth muscle contraction. The MAPK inhibitors PD098059 and UO126 both inhibited serotonin (5-HT) concentration-response relations more effectively in carotid arteries from term fetal lambs, than in corresponding arteries from mature non-pregnant adult sheep. This inhibition involved significant decreases in both the pD2 (adult: 2-fold; fetus: 4- to 15-fold) and the maximum efficacy (adult: 15-19%; fetus: 34-39%) of 5-HT. Accompanying this age-dependent effect on contraction, quantitative Western blot assays revealed that ERK1 and ERK2 abundances were 39% and 164% greater, respectively, in fetal than in adult carotid arteries. The abundance of the putative ERK target, caldesmon, however, was about 7-fold greater in adult than in fetal arteries. Together, the present results support the view that ERK abundance and activity is upregulated in fetal relative to adult arteries, and that one consequence of this upregulation is that the contribution of ERKs to contraction, at least that initiated by 5-HT2a receptors, is greater in fetal than adult carotid arteries. Whereas the phosphorylation mechanisms through which ERKs augment contraction remain uncertain and controversial, the present results suggest that emphasis should be shifted away from caldesmon and toward other critical contractile proteins, and how these proteins may contribute differently to development of agonist-induced contractile force in immature and mature arteries.