Total Recall: Can We Reshape T Cell Memory by Lymphoablation?

Despite recent advances in immunosuppression, donor-reactive memory T cells remain a serious threat to successful organ transplantation. To alleviate damaging effects of preexisting immunologic memory, lymphoablative induction therapies are used as part of standard care in sensitized recipients. However, accumulating evidence suggests that memory T cells have advantages over their naive counterparts in surviving depletion and expanding under lymphopenic conditions. This may at least partially explain the inability of existing lymphoablative strategies to improve long-term allograft outcome in sensitized recipients, despite the well-documented decrease in the frequency of early acute rejection episodes. This minireview summarizes the insights gained from both experimental and clinical transplantation as to the effects of existing lymphoablative strategies on memory T cells and discusses the latest research developments aimed at improving the efficacy and safety of lymphoablation.

Dieback of Pinus nigra Seedlings Caused by a Strain of Trichoderma viride.

Four different fungi (Trichoderma viride, T. harzianum, Phomopsis sp., and Mortierella sp.) were isolated from 6-year-old Pinus nigra plants showing stunting and high incidence of mortality in a reforestation area of the National Park of Abruzzo, Lazio, and Molise (central Italy). Tests conducted on P. nigra revealed the pathogenic behavior of T. viride isolates with 30 to 80% mortality in artificially inoculated 2-year-old seedlings. The pathogenicity of these isolates was also observed in 10-year-old P. nigra trees and on lemon fruit. This result, in agreement with the constant isolation of T. viride from diseased plants, suggests the possible role of this fungus in the decline of P. nigra plants. T. harzianum and two reference isolates of T. viridarium and T. trixiae did not cause any symptoms, while Phomopsis sp. and Mortierella sp. caused limited necroses around the inoculation point in a few seedlings. Their role in the decline of P. nigra seedlings was considered irrelevant. According to phylogenetic analyses, pathogenic isolates of T. viride clustered in a very uniform group containing strains from different geographic origin and hosts, but none previously reported as a biocontrol agent.

Haemodynamic determinants of the mitral valve closure sound: a finite element study.

Automatic acoustic classification and diagnosis of mitral valve disease remain outstanding biomedical problems. Although considerable attention has been given to the evolution of signal processing techniques, the mechanics of the first heart sound generation has been largely overlooked. In this study, the haemodynamic determinants of the first heart sound were examined in a computational model. Specifically, the relationship of the transvalvular pressure and its maximum derivative to the time-frequency content of the acoustic pressure was examined. To model the transient vibrations of the mitral valve apparatus bathed in a blood medium, a dynamic, non-linear, fluid-coupled finite element model of the mitral valve leaflets and chordae tendinae was constructed. It was found that the root mean squared (RMS) acoustic pressure varied linearly (r2= 0.99) from 0.010 to 0.259 mmHg, following an increase in maximum dP/dt from 415 to 12470 mm Hg s(-1). Over that same range, peak frequency varied non-linearly from 59.6 to 88.1 Hz. An increase in left-ventricular pressure at coaptation from 22.5 to 58.5mm Hg resulted in a linear (r2= 0.91) rise in RMS acoustic pressure from 0.017 to 1.41mm Hg. This rise in transmitral pressure was accompanied by a non-linear rise in peak frequency from 63.5 to 74.1 Hz. The relationship between the transvalvular pressure and its derivative and the time-frequency content of the first heart sound has been examined comprehensively in a computational model for the first time. Results suggest that classification schemes should embed both of these variables for more accurate classification.

Dynamic finite element implementation of nonlinear, anisotropic hyperelastic biological membranes.

We present a novel method for the implementation of hyperelastic finite strain, non-linear strain-energy functions for biological membranes in an explicit finite element environment. The technique is implemented in LS-DYNA but may also be implemented in any suitable non-linear explicit code. The constitutive equations are implemented on the foundation of a co-rotational uniformly reduced Hughes-Liu shell. This shell is based on an updated-Lagrangian formulation suitable for relating Cauchy stress to the rate-of-deformation, i.e. hypo-elasticity. To accommodate finite deformation hyper-elastic formulations, a co-rotational deformation gradient is assembled over time, resulting in a formulation suitable for pseudo-hyperelastic constitutive equations that are standard assumptions in biomechanics. Our method was validated by comparison with (1) an analytic solution to a spherically-symmetric dynamic membrane inflation problem, incorporating a Mooney-Rivlin hyperelastic equation and (2) with previously published finite element solutions to a non-linear transversely isotropic inflation problem. Finally, we implemented a transversely isotropic strain-energy function for mitral valve tissue. The method is simple and accurate and is believed to be generally useful for anyone who wishes to model biologic membranes with an experimentally driven strain-energy function.

Phytophthora palmivora a New Pathogen of Lavender in Italy.

Root rot caused by Phytophthora nicotianae is considered the most serious disease of lavender in commercial cultivations in Italy. In summer 2001, in the Gela area (Sicily), ≈60% of 34,000 2-year-old landscape shrubs of English lavender (L. angustifolia) grown in a clay loam soil showed symptoms of dieback associated with root rot. Plants had been transplanted from pots in May and watered using a trickle irrigation system. A species of Phytophthora was isolated consistently from roots of symptomatic plants using potato dextrose agar (PDA) containing benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol. The species was identified as P. palmivora on the basis of morphological and cultural characters. Ten representative single-zoospore isolates were characterized. On agar media, the isolates produced elliptical to ovoid, papillate sporangia, with a mean length/breadth ratio of 1.8. Sporangia, produced on sporangiophores forming simple sympodia (as many as 20 sporangia per sympodium), were caducous with a short pedicel (mean pedicel length = 5 µm) and a conspicuous basal plug. In addition to typical sporangia, all isolates produced sporocysts, i.e., subglobose, nonpapillate sporangia (2). The minimum temperature for mycelium growth on PDA was 10°C, the optimum was 27°C, and the maximum was 35°C. All isolates were A1 mating type. Antheridia were amphyginous. The identification was confirmed by electrophoresis of mycelial proteins on a polyacrylamide slab gel. Electrophoretic banding patterns of total soluble proteins and eight isozymes of the isolates from lavender were identical to those of a reference isolate of P. palmivora from olive (1). Conversely, the electrophoretic phenotype of the isolates from lavender was distinct from those of reference isolates of other species, including P. cactorum, P. capsici, P. citrophthora, P. nicotianae, and P. tropicalis. The pathogenicity of a representative isolate of P. palmivora from lavender was tested in the greenhouse using 6-month-old plants of English lavender, Rosea, a commercial cultivar very susceptible to root rot caused by P. nicotianae (3). Inoculum was produced on a mixture of vermiculite and autoclaved oat seeds (4) and mixed with soil (sand/lime/peat 1:1:1) at a concentration of 4% (vol/vol). Plants were transplanted to pots filled with infested soil. Control plants were grown in pots containing noninfested soil. After transplanting, all pots were flooded for 24 h by plugging the drain hole. Three months after transplanting all plants grown in pots containing infested soil showed extensive root necrosis and dieback symptoms. Control plants remained healthy. P. palmivora was recovered from roots of symptomatic plants. To our knowledge, this is the first report from Italy of P. palmivora on lavender. Root rot caused by P. palmivora may be a potential problem for commercial cultivation of lavender. References: (1) S. O. Cacciola et al. Plant Dis. 84:1153, 2000. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St Paul, MN, 1996. (3) G. Minuto et al. Inf. Fitopatol. 51:69, 2001. (4) E. Sánchez-Hernández et al. Plant Dis. 85:411, 2001.

Comparison of effortful and noneffortful swallows in healthy middle-aged and older adults.

OBJECTIVE: To assess the effects of effortful swallowing, a common compensatory strategy for dysphagia, on the bolus and swallowing mechanism of middle-aged and older men and women. DESIGN: Case-controlled design in which subjects completed both the intervention technique and the control behavior. SETTING: A university hospital. PARTICIPANTS: Sixty-four healthy men and women between 45 and 93 years of age from the community. INTERVENTIONS: Participants swallowed 3-mL thin liquid boluses both normally and using the effortful swallow strategy. MAIN OUTCOMES MEASURES: The biomechanics and bolus flow patterns of swallows were analyzed from videofluoroscopic and simultaneous oral pressure data. RESULTS: Subjects at all ages generated significantly increased oral pressures at each sensor location using the effortful swallow (p = .0001), with the pressure increase greater for the middle-aged subjects compared with older subjects. Several durational measures were significantly longer with the effortful swallow including: hyoid maximum anterior excursion (p < .04), laryngeal vestibule closure (p < .0001), and duration of the upper esophageal sphincter opening (p =.0001). The hyoid bone moved further in the superior direction with the effortful swallow (p = .002). There was a trend of decreased oral residue with the effortful swallow (p = .06). CONCLUSION: Biomechanical and bolus flow aspects of swallowing changed when healthy individuals performed effortful swallows with 3-mL boluses.

The fluid mechanics of bolus ejection from the oral cavity.

The squeezing action of the tongue against the palate provides driving forces to propel swallowed material out of the mouth and through the pharynx. Transport in response to these driving forces, however, is dependent on the material properties of the swallowed bolus. Given the complex geometry of the oral cavity and the unsteady nature of this process, the mechanics governing the oral phase of swallowing are not well understood. In the current work, the squeezing flow between two approaching parallel plates is used as a simplified mathematical model to study the fluid mechanics of bolus ejection from the oral cavity. Driving forces generated by the contraction of intrinsic and extrinsic lingual muscles are modeled as a spatially uniform pressure applied to the tongue. Approximating the tongue as a rigid body, the motion of tongue and fluid are then computed simultaneously as a function of time. Bolus ejection is parameterized by the time taken to clear half the bolus from the oral cavity, t(1/2). We find that t(1/2) increases with increased viscosity and density and decreases with increased applied pressure. In addition, for low viscosity boluses (mu approximately 100 cP), density variations dominate the fluid mechanics while for high viscosity boluses (mu approximately 1000 cP), viscosity dominates. A transition region between these two regimes is found in which both properties affect the solution characteristics. The relationship of these results to the assessment and treatment of swallowing disorders is discussed.

Local longitudinal muscle shortening of the human esophagus from high-frequency ultrasonography.

We analyzed local longitudinal shortening by combining concurrent ultrasonography and manometry with basic principles of mechanics. We applied the law of mass conservation to quantify local axial shortening of the esophageal wall from ultrasonically measured cross-sectional area concurrently with measured intraluminal pressure, from which correlations between local contraction of longitudinal and circular muscle are inferred. Two clear phases of local longitudinal shortening were observed during bolus transport. During luminal filling by bolus fluid, the muscle layer distends and the muscle thickness decreases in the absence of circular or longitudinal muscle contraction. This is followed by local contraction, first in longitudinal muscle, then in circular muscle. Maximal longitudinal shortening occurs nearly coincidently with peak intraluminal pressure. Longitudinal muscle contraction begins before and ends after circular muscle contraction. Larger longitudinal shortening is correlated with higher pressure amplitude, suggesting that circumferential contractile forces are enhanced by longitudinal muscle shortening. We conclude that a peristaltic wave of longitudinal muscle contraction envelops the wave of circular muscle contraction as it passes through the middle esophagus, with peak longitudinal contraction aligned with peak circular muscular contraction. Our results suggest that the coordination of the two waves may be a physiological response to the mechanical influence of longitudinal shortening, which increases contractile force while reducing average muscle fiber tension by increasing circular muscle fiber density locally near the bolus tail.

Heterologous transposition in Aspergillus nidulans.

Aspergillus nidulans is one of the model ascomycete fungi. Transposition events have never been described in this organism. We have determined that this organism has at least 13 copies of a Fot1-related element. These copies are transcribed, non-methylated and polymorphic in various wild isolates. In spite of this, we have failed to isolate transposon insertions when the resident niaD gene is used as a transposon trap. This contrasts with the situation described previously in Fusarium oxysporum. We show that two elements of F. oxysporum, Fot1 and impala, transpose efficiently in A. nidulans. We have developed the impala system by tagging it with the yA gene. This permits the visual detection of the transposon by the colour of the conidiospores. We demonstrate that no endogenous transposase of A. nidulans is able to act in trans on a defective impala element, whereas its own transposase driven by two different promoters is able to mobilize this element. The frequency of excision of these modified elements is between 10(-4) and 10(-5). Loss of the transposable element occurs in about 10% of all excision events. In the remaining 90%, the transposon seems to be integrated at random positions in the genome. The availability of mitochondrially inherited mutations has allowed us to demonstrate that hybrid dysgenesis is apparently absent in A. nidulans. The development of this system opens the way to investigating the mechanism underlying the paucity of transposition events leading to visible phenotypes. It should allow us to develop efficient gene-tagging tools, useful in this and other fungi.

First Report of Phytophthora citrophthora Causing Fruit Brown Rot of Feijoa in Italy.

Feijoa (Feijoa sellowiana) is native to South America. In the early 20th Century it was introduced into Sicily (southern Italy), where it is grown as an ornamental plant and for its fruits. In 1985 a Phytophthora brown rot of feijoa fruits was reported in the province of Syracuse (eastern Sicily) (2). Several species of Phytophthora, including P. citricola, P. citrophthora, and P. nicotianae, were recovered from soil samples taken from trees with infected fruits. These species were experimentally inoculated on detached feijoa fruits and all incited symptoms of brown rot. However, only P. citricola was isolated from naturally infected fruits. In early autumn 1999, an outbreak of Phytophthora brown rot of feijoa fruits was observed in the Syracuse province, in the same site where the disease had been first recorded. P. citricola (95% of the isolates) and P. citrophthora (5% of the isolates) were recovered from symptomatic fruits. The species were identified on the basis of morphological and cultural characters according to Erwin and Ribeiro (1). The P. citricola isolates formed colonies with a distinctive chrysanthemum pattern on potato-dextrose agar (PDA), had an optimum temperature for radial growth of 25°C, and were homothallic with paragynous antheridia and spherical oogonia (mean diameter of oogonia= 20 µm). Sporangia, which were produced only in water or saline solution, were semi-papillate (often with two apices) and variable in shape. The P. citrophthora isolates formed petaloid colonies on PDA, had an optimum temperature of 25°C, and produced noncaducous, papillate (frequently bipapillate), ovoid to limoniform sporangia. They did not produce gametangia. The identification of both species was confirmed by the electrophoresis of mycelial proteins on polyacrylamide slab gel. The electrophoretic patterns of total proteins and four isozymes (alkaline phosphatase, esterase, malate dehydrogenase, and superoxide dismutase) of the P. citricola and P. citrophthora isolates from feijoa were identical to those of reference isolates of these two species from various other hosts. Conversely, they were clearly distinct from the electrophoretic patterns of reference isolates of P. cactorum, P. capsici, P. nicotianae, and P. palmivora. The random amplified polymorphic DNA patterns of the P. citrophthora isolates from feijoa obtained by polymerase chain reaction (RAPD-PCR) were compared with those of reference isolates of other species of Phytophthora and those of P. citrophthora isolates obtained from citrus trees with symptoms of trunk gummosis and root rot, grown in the immediate vicinity of feijoa trees. DNA was extracted and analyzed following previously described procedures, using 16 decamer primers (3). The RAPD-PCR patterns of the P. citrophthora isolates from feijoa were identical to those of the isolates from citrus but were distinct from those of reference isolates of the other species of Phytophthora, suggesting that inoculum of P. citrophthora may have originated from infected citrus trees. P. citricola is known as a causal agent of fruit brown rot of feijoa and guava (Psidium guajava), a closely related species (1). Conversely, this is the first report of natural infections of P. citrophthora on feijoa fruits. References: (1) D. C. Erwin and O. K. Ribeiro, 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society. St. Paul, MN. (2) G. Magnano di San Lio and R. Tuttobene. Inf. Fitopatol. 85:43, 1985. (3) Q. Migheli et al. Eur. J. Plant Pathol. 104:49, 1998.

Age effects on the temporal evolution of isometric and swallowing pressure.

BACKGROUND: The tongue plays a key role in bolus propulsion through the oropharyngeal chamber. In this study, possible age effects on the magnitude and timing of lingual pressure generation were analyzed. METHODS: Oral pressure was measured during isometric and swallowing tasks for 10 elderly (mean age = 81 years) and 10 young (mean age = 51 years) subjects. Three trials each of the isometric task and swallows of three different boluses (3 ml semisolid, 3 ml liquid, and 10 ml liquid) were performed by each subject. The timing and magnitude of isometric and swallowing pressure generation along with the pattern of the swallowing pressure waveform were analyzed. RESULTS: Whereas maximum lingual isometric pressures decreased with age (p < .001). no significant age difference was found for swallowing pressure. Time taken to reach peak pressure also was reduced with age in both the isometric task and swallows of liquid boluses (p < .05), while no significant age effect was found for semisolid swallows. Finally, only elderly subjects showed a pattern of liquid swallowing pressure generation in which multiple lingual gestures were required to reach peak pressure (termed "pressure building"), a pattern demonstrated by both young and elderly groups for semisolids. CONCLUSIONS: Decreased lingual strength with age combined with unchanging swallowing pressure leads to a decreased "pressure reserve," perhaps leaving older individuals more at risk for dysphagia resulting from insults directly or indirectly to the swallowing system. Additionally, swallowing is generally "slowed" with age, apparently due to both central and peripheral factors, and this change may have an impact on bolus flow outcomes.

The HSV-1 2-kb latency-associated transcript is found in the cytoplasm comigrating with ribosomal subunits during productive infection.

We have examined the nuclear and cytoplasmic distribution of the latency-associated transcripts (LATs) of HSV-1. During latency these transcripts accumulate in the nuclei of neurons in the peripheral and central nervous system of infected animals. However, our Northern blot analyses demonstrate that the 2-kb LAT is found in the cytoplasm of HSV-1-infected CV-1 cells, and brainstems of HSV-1 productively infected mice. Like the nuclear LAT from latently infected tissue, most of the cytoplasmic 2-kb LAT from lytically infected CV-1 cells is unpolyadenylated. In order to determine if cytoplasmic LAT could be translated, we compared its distribution with that of glycoprotein C mRNA in polysome profiles from HSV-1-infected tissue culture cells. Specific association of RNAs to polysomes was verified by disruption of polysomes with EDTA or puromycin. Analyses of numerous experiments indicate that most of the cytoplasmic 2-kb LAT migrates at the position of ribosomal subunits in polysome profiles. Thus, the 2-kb LAT may not be efficiently translated during productive infection. This suggests that if the 2-kb LAT is indeed translated, its translation may be tightly regulated during HSV-1 infection, possibly in a cell type- or cell cycle-specific manner. Another possibility is that the 2-kb LAT is not a translated RNA but may have another function, possibly related to translation as indicated by its apparent association to ribosomal complexes.

Herpes simplex virus type 1 latency-associated transcript (LAT) promoter deletion mutants can express a 2-kilobase transcript mapping to the LAT region.

The results of studies in several laboratories suggest that a TATA box-containing promoter located in the herpes simplex virus type 1 internal long repeat and long terminal repeat elements drives expression of the latency-associated transcripts (LATs). In the present study, we show that expression of a 2-kb LAT-related transcript can occur in the absence of this LAT TATA promoter, indicating the existence of a cryptic promoter. By Northern (RNA) blot analysis, we have examined LAT expression by herpes simplex virus type 1 variant strains KOS/29 and 1704, which contain deletions of the LAT promoter region. Our data indicate that KOS/29, despite lacking the 203-bp fragment which contains the LAT TATA box, can express a 2-kb LAT-related transcript during productive infection in tissue culture and in mouse trigeminal ganglia during acute infection and reactivation. Similarly, strain 1704, which contains a larger deletion in this promoter region, also expresses a 2-kb LAT-related transcript during tissue culture infection and reactivation of latently infected trigeminal ganglia. However, LATs are not expressed with either virus during latency. Northern blot analysis with a single-stranded, oligonucleotide probe demonstrates that the 2-kb LAT and LAT-related transcript are colinear and share a large area of sequence similarity. These findings suggest the existence of a second promoter in the LAT gene which can function during lytic infection and reactivation, at least in the absence of the LAT TATA promoter. We propose that this cryptic promoter is located either in a proximal region approximately 300 bp upstream of the start site of the 2-kb LAT or in a distal region starting over 1,226 bp upstream of this site.

An HSV-1 mutant lacking the LAT TATA element reactivates normally in explant cocultivation.

In order to examine if mutations within the LAT promoter region of HSV-1 are sufficient to change the reactivation phenotype, a mutant (KOS/29) containing a deletion of the LAT TATA element was used to establish latent infections in mouse ganglia by corneal inoculation. During the acute phase of infection, KOS/29 replicated as efficiently as its wild-type parent. As previously noted, latent KOS/29 infections were totally devoid of LAT gene transcripts (Dobson A. T., Sederati F., Devi-Rao G., Flanagan J., Farrell M. J., Stevens J. G., Wagner E. K., and Feldman L. T., J. Virol. 63, 3844-3851 (1989))). However, unlike other null mutants, KOS/29 reactivated from explanted ganglia with a kinetics similar to that of the LAT competent parent. These data show that the deletion created in KOS/29, removing the LAT TATA promoter element and small upstream and downstream flanking sequences, is not enough to alter the reactivation phenotype and that efficient reactivation can occur in the absence of any detectable LAT expression during latency.

Herpes simplex virus type 1 mutant strain in1814 establishes a unique, slowly progressing infection in SCID mice.

Ocular infection of immunocompetent (BALB/c) mice with wild-type herpes simplex virus type 1 (HSV-1) 17+ may lead to acute fatal encephalitis; however, in surviving animals, a latent (nonproductive) infection of the nervous system is established. In contrast, 17+ infection invariably kills mice with severe combined immunodeficiency (SCID mice) within 2 weeks. Ocular infection of immunocompetent mice with a mutant HSV-1 strain, in1814, which does not produce a functional alpha-transinducing protein, results in no detectable viral replication in the nervous system during the time corresponding to the acute phase of infection, no mortality, and the establishment of latency. In SCID mice, however, the in1814 virus establishes a unique, slowly progressing infection. In studying the courses of in1814 infection in SCID and BALB/c mice, we found that although intact B- and/or T-lymphocytic functions were required for the control of viral replication in the nervous system, some of the infected neurons of SCID mice seemed to be able to restrict in1814 replication and harbor the virus in a latent state.

Apolipoprotein-E messenger RNA in rat ovary is expressed in theca and interstitial cells and presumptive macrophage, but not in granulosa cells.

Apolipoprotein-E (apoE) is a constituent of various lipoproteins and is a ligand for cellular lipoprotein receptors. Unlike most apolipoproteins, apoE is synthesized in peripheral tissues, including those engaged in steroidogenesis. ApoE expression in adrenal cells inhibits cholesterol utilization for steroid synthesis and blocks signal transduction via the protein kinase-A pathway. In cultured ovarian thecal/interstitial cells, exogenous apoE has been shown to inhibit LH-induced androgen synthesis. These findings support a role for apoE as an autocrine or paracrine factor involved in regulating steroidogenesis. In the present study in situ hybridization was used to identify cell types that express apoE mRNA in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with PMSG to stimulate follicular development, and from PMSG-treated rats that were subsequently administered hCG to stimulate ovulation and luteinization. ApoE mRNA was localized to theca and interstitial cells of follicles in animals at all stages of the estrous cycle as well as in immature rats treated with PMSG. ApoE mRNA was not detected in oocytes, cumulus cells, or granulosa cells. High levels of apoE mRNA also were expressed by localized clusters of presumptive macrophages in atretic follicles and degenerating corpora lutea. This complex pattern of expression may indicate that apoE has multiple functions in the rat ovary. ApoE made by theca and interstitial cells may act locally as an autocrine factor to regulate androgen production. ApoE made in atretic follicles and regressing corpora lutea may serve to facilitate local transport and reutilization of lipid released as these structures degenerate.

Differential regulation of apolipoprotein-E messenger RNA in zona fasciculata cells of rat adrenal gland determined by in situ hybridization.

Previous studies showed that apolipoprotein-E (apoE) mRNA is regulated in rat adrenal gland by treatments that alter adrenal gland cholesterol content and steroidogenesis. In the present study cell types expressing apoE mRNA were determined by in situ hybridizations using an [alpha-35S]UTP-labeled RNA probe. Autoradiographic grains were counted to compare apoE expression in adrenal glands from control and experimentally treated animals. In control adrenal gland, zona (z.) fasciculata and z. reticularis exhibited the highest level of apoE mRNA expression, with lower levels in z. glomerulosa and medulla. Dexamethasone (DEX) treatment selectively increased apoE mRNA 3-fold in outer z. fasciculata, but not in other adrenal zones. ApoE mRNA expression appeared to be lower in adrenal glands from 4-aminopyrazolopyrimidine-treated rats, in that differences among adrenal gland zones were abolished. DEX treatment increased adrenal gland cholesteryl ester and oil red O staining in z. fasciculata cells in which the apoE mRNA concentration was increased as well as in other cortical cells in which apoE mRNA was unchanged. Aminoglutethimide administration led to a large increase in oil red O staining throughout the cortex, including z. fasciculata, without affecting apoE mRNA expression. These data suggest that adrenal gland apoE mRNA expression is not closely coupled to cellular cholesterol concentrations. Increased apoE mRNA expression in z. fasciculata of DEX-treated animals suggests an inverse relationship between apoE mRNA concentration and the level of steroidogenesis. This result is consistent with the proposal that apoE may play a role in regulating the utilization of cholesterol for steroid production.

Relationship between apolipoprotein E mRNA expression and tissue cholesterol content in rat adrenal gland.

Among extrahepatic tissues the adrenal gland has one of the highest concentrations of apoE mRNA and the highest rate of apoE synthesis. In the present investigation several previously described in vivo treatments were used to assess the relationship between apoE expression and cellular cholesterol in the rat adrenal gland. Treatment of rats with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) to lower serum cholesterol concentration and deplete adrenal gland cholesterol content decreased adrenal gland apoE mRNA concentration. These adrenal responses were blocked by dexamethasone (DEX) suggesting that the effect of 4-APP occurred indirectly via stimulation of the adrenal gland by endogenous adrenocorticotrophic (ACTH). Relative to control rats, DEX treatment increased both adrenal gland cholesterol content and apoE mRNA concentration. Concurrent ACTH and DEX administration reduced both adrenal gland cholesterol content and apoE mRNA concentration relative to DEX-treated rats. ACTH administration also rapidly decreased adrenal gland apoE mRNA concentration and cholesterol content in rats pretreated with DEX. In all the above experiments, adrenal gland cholesterol content and apoE mRNA concentration were positively correlated (r = 0.78, P = 0.0001). In contrast, aminoglutethimide treatment, which blocks adrenal gland steroidogenesis and greatly increases adrenal gland cholesterol content, was without effect on apoE mRNA concentration. ACTH administration to rats treated with DEX + aminoglutethimide resulted in decreased adrenal apoE mRNA despite greatly increased adrenal cholesterol content. This uncoupling of adrenal gland cholesterol content and apoE mRNA concentration suggests that apoE mRNA expression and cellular cholesterol are regulated independently by ACTH.

Estrogen-induced destabilization of yolk precursor protein mRNAs in avian liver.

In addition to regulating gene expression at the level of transcription, estrogen is generally considered to selectively stabilize induced mRNAs against degradation. As a result of mRNA stabilization, estrogen-induced mRNAs accumulate to much higher levels in target cells, and the encoded proteins are made at much greater rates than would occur on the basis of transcriptional activation alone. The present study examined the effect of estrogen on the stabilities of avian liver mRNAs that code for the yolk precursor proteins apolipoprotein (apo) II and vitellogenin (VTG) II. The results show that the degradation rates of apoII and VTG II mRNAs during hormone withdrawal are dramatically altered by the duration of prior estrogen treatment. During the 2 days required for the hormonal inductions of these mRNAs to new steady states, the turnover rates of both mRNAs were the same in the presence and absence of estrogen (t1/2 = 13 h). This result indicates that mRNA stabilization does not contribute to the extensive accumulation of apoII and VTG II mRNAs. When the duration of estrogen treatment was extended beyond 3 days, however, hormone withdrawal led to the rapid (t1/2 = 1.5 h) and selective destabilization of these mRNAs. This result suggests that estrogen induced a destabilization activity that was only functional following hormone withdrawal. Thus, the point at which estrogen alters mRNA stability is at the level of mRNA degradation. An absence of detectable apoII mRNA degradation intermediates during either the slow or rapid mode of mRNA decay suggests that the rate-limiting step for apoII mRNA turnover is an estrogen-sensitive targeting event that marks the mRNA for rapid degradation.

Regulation of chicken apolipoprotein B: cloning, tissue distribution, and estrogen induction of mRNA.

Apolipoprotein (apo) B is a major protein component of plasma very low-density and low-density lipoproteins (VLDL and LDL, respectively) and serves as a recognition signal for the cellular binding and internalization of LDL by the apoB/E receptor. In contrast to the situation in mammals, avian apoB is also a component of specialized VLDL particles that are produced by the liver in response to estrogen. These particles transport cholesterol and triglyceride from the liver to the ovary for deposition in egg yolk. We report here the identification and characterization of cDNA clones for chicken apoB and their use in examining the tissue distribution and hormonal regulation of chicken apoB mRNA. The cDNA clones were identified by immunological screening of a phage lambda gt11 library constructed with hen liver mRNA and their identity was supported by sequence comparisons with mammalian apoB. The chicken apoB mRNA is approximately the same size as mammalian apoB mRNA (14 kb), and, as occurs in mammals, is present at high levels in liver and small intestine. Unlike mammals, the chicken apoB mRNA is also found at high levels in the kidney, consistent with previous protein biosynthetic studies. A DNA-excess solution-hybridization assay was used to quantitate apoB mRNA in these tissues and to examine its hormonal regulation. In control roosters the liver and kidney contained 65% and 10%, respectively, as much apoB mRNA as the small intestine. Within 24 h after estradiol administration, apoB mRNA was increased five- to seven-fold in liver but was unchanged in intestine and kidney. The increase in apoB mRNA content and the kinetics of induction parallel hepatic apoB synthesis, indicating that estrogen regulates apoB production through changes in the cellular abundance of apoB mRNA. The apoB mRNA increased rapidly following hormone treatment while the mRNA for another VLDL protein (apoII) showed a lag or slow phase of several hours before significant mRNA accumulation occurred. These data indicate that the liver can respond immediately to estrogen to increase apoB mRNA accumulation, while apoII mRNA accumulation appears to involve additional events or signals which occur slowly and are specific to this gene.