Evaluation of the size of the microsphere loss from the myocardium in rats in a chronic experiment

The loss of radioactive microspheres (15 microns in diameter) from the rat myocardium was investigated during chronic experiment. Microspheres were injected to donor rats, the hearts were removed 20-30 min later and the number of microspheres trapped in the heart was determined on gamma-counter. After that the hearts were transplanted into the abdominal cavity of recipient rats using microsurgery technique. 5-24 days later the recipients were killed, the transplanted hearts were removed and radioactivity was measured again. It was shown that not more than 10% of microspheres were lost from the myocardium over a 5-24-day period, with the value of microsphere loss independent of the time period.     

Effects of hypophysectomy, growth hormone, and thyroxine on protein turnover in heart.

Cardiac atrophy following hypophysectomy was accompanied by decreased heart content of RNA and polysomes and increased levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in vitro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower RNA content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, heart RNA content and rates of protein synthesis were equal to or greater than those found in normal hearts. When plasma levels of amino acids, glucose, fatty acids, and insulin, and rates of beating and ventricular pressure development observed in normal and hypophysectomized rats were simulated during in vitro perfusion, hearts from hormone-deficient rats had reduced rates of protein synthesis but unaltered rates of degradation. Cathepsin D activity in heart homogenates (+ Triton X-100) was elevated during cardiac atrophy when expressed per g of tissue but not when expressed per heart.     

The coronary vasodilator mediator released by hypoxia and isoprenaline is not affected by cyclo-oxygenase inhibition

Donor and recipient guinea-pig hearts were perfused in series, the recipient being perfused with regassed donor effluent. Coronary perfusion pressure and force and rate of contraction were measured. Exposure of donor hearts to hypoxia (1.5 min) and to isoprenaline (5 ng) caused the appearance of vasodilator material in recipient hearts, the direct beta-adrenoceptor effects of isoprenaline carried over in the effluent being antagonized in the recipient by propranolol. Cyclo-oxygenase was inhibited by infusion of meclofenamate (60 micrograms X min-1) which consistently abolished the vasodilator responses to arachidonic acid added to the donor. The vasodilator responses of the donor to hypoxia and isoprenaline were unaffected by meclofenamate. The falls in perfusion pressure of the recipient in response to material released by these procedures were also not significantly different before (hypoxia, 11.5 +/- 2.6mm Hg; isoprenaline, 10.3 +/- 1.3mm Hg) and during the infusion (hypoxia, 10.2 +/- 4.1; isoprenaline, 11.0 +/- 1.3mm Hg). The coronary vasodilator responses to hypoxia and isoprenaline and the vasodilator material released by these procedures do not therefore appear to be due to products of arachidonic acid via cyclo-oxygenase pathways. Furthermore, since there was also no potentiation of the responses, there does not appear to be a concomitant release of a prostanoid to inhibit the major vasodilator material. Adenosine, as the likely candidate for this predominant vasodilator mediator, is discussed.     

The effects of lactate, acetate, glucose, insulin, starvation and alloxan-diabetes on protein synthesis in perfused rat hearts.

Compared with glucose, lactate + acetate stimulated ventricular protein synthesis in anterogradely perfused hearts from fed or 72 h-starved rats. Stimulation was greater on a percentage basis in starved rats. Atrial protein synthesis was not detectably stimulated by lactate + acetate. Insulin stimulated protein synthesis in atria and ventricles. The stimulation of protein synthesis by lactate + acetate and insulin was not additive, the percentage stimulation by insulin being less in the ventricles of lactate + acetate-perfused hearts than in glucose-perfused hearts. Perfusion of hearts from 72 h-starved or alloxan-diabetic rats with glucose + lactate + acetate + insulin did not increase protein-synthesis rates or efficiencies (protein synthesis expressed relative to total RNA) to values for fed rats, implying there is a decrease in translational activity in these hearts. In the perfused heart, inhibition of protein synthesis by starvation and its reversal by re-feeding followed a relatively prolonged time course. Synthesis was still decreasing after 3 days of starvation and did not return to normal until after 2 days of re-feeding.     

The coronary vasodilator mediator released by hypoxia and isoprenaline is not affected by cyclo-oxygenase inhibition

Donor and recipient guinea-pig hearts were perfused in series, the recipient being perfused with regassed donor effluent. Coronary perfusion pressure and force and rate of contraction were measured. Exposure of donor hearts to hypoxia (1.5 min) and to isoprenaline (5 ng) caused the appearance of vasodilator material in recipient hearts, the direct β-adrenoceptor effects of isoprenaline carried over in the effluent being antagonized in the recipient by propranolol. Cyclo-oxygenase was inhibited by infusion of meclofenamate (60μg.min⁻¹) wich consistently abolished the vasodilator responses to arachidonic acid added to the donor. The vasodilator responses of the donor to hypoxia and isoprenaline were unaffected by meclofenamate. The falls in perfusion pressure of the recipient in response to material released by these procedures were also not significantly different before (hypoxia, 11.5±2.6mm Hg; isoprenaline, 10.3±1.3mm Hg) and during the infusion (hypoxia, 10.2±4.1; isoprenaline, 11.0±1.3mm Hg). The coronary vasodilator responses to hypoxia and isoprenaline and the vasodilator material released by these procedures do not therefore appear to be due to products of arachidonic acid via cyclo-oxygenase pathways. Furthermore, since there was also no potentiation of the responses, there does not appear to be a concomitant release of a prostanoid to inhibit the major vasodilator material. Adenosine, as the likely candidate for this predominant vasodilator mediator, is discussed.     

Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats.

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.     

Spontaneous acceptance of liver transplants in rodents: evidence that liver leucocytes induce recipient T-cell death by neglect

In many animal models transplanted livers are not rejected, even when there is a complete MHC mismatch between the donor and recipient and the recipient is not immunosuppressed. This distinguishes liver transplants from other organs, such as kidneys and hearts, which are rapidly rejected in mismatched individuals. Acceptance of transplanted livers in a rat model is not due to the absence of an immune response to the liver and there is a rapid, abortive response that is ultimately exhausted. Donor leucocytes transferred with the liver appear to be responsible for both liver acceptance and the abortive activation of the recipient's T cells. The immune mechanism of liver transplant acceptance appears to be due to 'death by neglect' in which T cells are activated to express IL-2 and IFN-gamma mRNA in the recipient lymphoid tissues, but not at adequate levels within the graft. Subsequently the activated T cells die leading to specific clonal deletion of liver donor-reactive T cells. These findings have important implications for liver transplant patients as immunosuppressive drugs that are given to prevent rejection can also interfere with this form of tolerance. In addition, it might be possible to modify the immunosuppressive drug treatment of transplant patients to promote the process of death by neglect of recipient alloreactive T cells.     

Transplantation of germ cells from rabbits and dogs into mouse testes.

Spermatogonial stem cells of a fertile mouse transplanted into the seminiferous tubules of an infertile mouse can develop spermatogenesis and transmit the donor haplotype to progeny of the recipient mouse. When testis cells from rats or hamsters were transplanted to the testes of immunodeficient mice, complete rat or hamster spermatogenesis occurred in the recipient mouse testes, albeit with lower efficiency for the hamster. The objective of the present study was to investigate the effect of increasing phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation. Testis cells were collected from donor rabbits and dogs and transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. In separate experiments, rabbit or dog testis cells were frozen and stored in liquid nitrogen or cultured for 1 mo before transplantation to mice. Recipient testes were analyzed, using donor-specific polyclonal antibodies, from 1 to >12 mo after transplantation for the presence of donor germ cells. In addition, the presence of canine cells in recipient testes was demonstrated by polymerase chain reaction using primers specific for canine alpha-satellite DNA. Donor germ cells were present in the testes of all but one recipient. Donor germ cells predominantly formed chains and networks of round cells connected by intercellular bridges, but later stages of donor-derived spermatogenesis were not observed. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh and cryopreserved germ cells can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion.     

Reduced adenosine release from the aged mammalian heart

Adenosine (ADO) released in the heart results in enhanced coronary blood flow and reduced catecholamine release and myocardial responsiveness to adrenergic stimulation (anti-adrenergic action). ADO release from the adrenergic-stimulated aged heart is less than that from the young adult heart. Because adrenergic signaling in the aged heart is impaired, this study was conducted to determine if reduced ADO release from the aged heart results from this reduced adrenergic responsiveness. Hearts of 3-4 months (young adult) and 21-22 months (aged) Fischer-344 rats were perfused with ADO deamination and re-phosphorylation inhibited. Coronary effluent ADO levels were determined. Cellular-free ADO levels with and without sodium acetate (NaAc)-induced mitochondrial AMP synthesis were assessed using formed S-adenosylhomocysteine (SAH) in L-homocysteine thiolactone (L-HC)-treated hearts. The activities of SAH-hydrolase were determined. Aged heart ADO release was 61% less than from young hearts. NaAc augmented young heart ADO release by 104%, while that of aged hearts remained unchanged. SAH synthesis was 51% and 56% lower in the aged heart in the absence and presence of NaAc, respectively, despite an 89% greater SAH hydrolase activity found in the aged hearts. Since synthesized AMP may be diverted to IMP and ultimately inosine by AMP deaminase, inosine release was determined. Aged heart inosine levels in the absence and presence of NaAc were 74% and 59% less than for the young hearts. It is concluded that a reduced mitochondrial AMP synthesis is in part responsible for the attenuation in ADO release from the adrenergic-stimulated aged heart.     

Super and half-sister discrimination by honey bee workers (Apis mellifera L.) in a trophallactic bioassay

Summary Kin recognition and nepotism between honeybee workers (Apis mellifera L.) was analysed in a trophallactic bio-assay. Donor workers were fed dyed sugar syrup and introduced into a recipient group consisting of 12 to 15 workers of the same colony. After allowing for 1 hour of trophallaxis, the distribution of the dyed food was analysed with spectrophotometry. The subfamily composition in the recipient group was varied such that the donor bees had to discriminate between workers of 2 to 7 different patrilines. Donor bees preferentially fed super sisters if few patrilines were present in the recipient group. However, preferential feeding was not observed if the recipient group consisted of workers of more than three subfamilies. Since the natural degree of polyandry causes intracolonial genetic variance to exceed the genetic variability in the experiments, nepotistic behaviour among workers may not reveal intranidal subfamily recognition in honeybees.     

Selection and procurement of hearts for transplantation.

The success of orthotopic heart transplantation depends wholly on satisfactory function of the new heart on completion of the operation. This in turn depends on the quality of the donor heart before its removal, the effectiveness of the methods used to preserve it during transport from the donor to the recipient hospital, and the accuracy of the operative procedure. From January 1979 to December 1983, 62 donor hearts were transplanted into 61 recipients at Papworth Hospital. These hearts were selected from 250 offered for consideration. The most common reasons for not proceeding with an initial inquiry were failure of the donor to meet the medical criteria for selection (77 cases) and lack of intensive care facilities or staff shortages such that a transplant could not be accommodated at the time of inquiry (80). Eight early deaths occurred, of which three were due to primary failure of the donor heart. Actual one and three year survivals for the whole programme were 58% and 50% respectively, the current actual one year survival being 70%. Forty per cent of patients selected for transplantation died while waiting for a heart to become available. Their average survival time was 46 days. The number of donor hearts referred for transplantation depends on public attitudes towards organ transplantation, the willingness of doctors looking after brain dead patients to seek permission from relatives for the heart to be donated, and the cooperation of local kidney transplant surgeons. A larger number of suitable donor hearts to choose from would enable more patients to be treated, as transplant operations could be arranged so that existing facilities were used to their maximum capacity.     

DNA synthesis in rat heart cells after injury and the regeneration of myocardia

The regenerative responses of the myocardia of post-natal rats of different age groups (1, 2, 3 and 4 weeks old) to an injury made by a clinical electricator were studied. DNA synthesis and the ultrastructural organization of the cardiac myocytes of the injured myocardia were examined for an evaluation of the potential for regeneration of the developing myocardia. The maximum labeling index of cardiac myocytes was observed in 1-week-old rats showing 8% labeled myocytes 3 days after injury as opposed to 3.2, 2.2 and 0.2% indices in 2-, 3- and 4-week-old rats respectively, 3 days after injury. In subsequent days after injury the labeling indices declined considerably in all age group hearts, and attained values less than 1% labeled myocytes 30 days after injury with the lowest labeling index in the oldest age group heart. When DNA synthesis in uninjured myocardial tissue adjacent to the injured tissue was examined, it was found to be significantly lower than it was in the injured tissue. However, both injured and adjacent uninjured tissue attained a peak in the labeling indices 3 days after injury, with the exception of 3- and 4-week-old uninjured tissue. The overall incorporation of ³H-thymidine into the DNA of heart cells as revealed by scintillation counts showed that the rate of incorporation of the isotope in younger hearts was significantly higher than in the older hearts. Non-muscle cells contributed significantly to the rise of scintillation counts in hearts of all age groups.Ultrastructural analyses of 1- to 4-week-old hearts showed that 24 hr after injury, injured areas of myocardia were heavily crowded with macrophages that surrounded damaged myocytes. Later on, fibroblasts and other non-muscle cells predominated the injury sites along with fibrous connective tissue. Scattered regenerating cardiac myocytes were frequently observed in the injury sites of 1- and 2-week-old hearts 3 days after injury. Myocytes were rare in the corresponding regions of 3- and 4-week-old hearts. Instead abundant non-muscle cells and fibrous connective tissue were predominant. In the fourth and final week of this study, the repaired areas of myocardia in 1- and 2-week-old rats contained more myocytes than those of the 3- and 4-week-old rats, and the repaired zone of the 1-week-old heart contained more myocytes than the repaired areas of the other age groups. These findings suggest that the mammalian myocardia possess an age-dependent potential for regeneration that involves the healing of injury sites with contractile and connective tissues.     

Gene conversion of immunoglobulin variable regions in mutagenesis cassettes by replacement PCR mutagenesis.

A technique, Replacement PCR Mutagenesis, was developed to replace one immunoglobulin variable region (V) in a M13 phage cassette with a different, homologous V. This allows the use of the same mutagenesis and subsequent expression vectors for many V regions or V segments. The method combines PCR of V fragments and in vitro mutagenesis. Primers homologous to 3' and 5' ends of both V regions initiate PCR synthesis of the V DNA fragment (donor) that will replace the V region (recipient) in M13. Donor V PCR DNA may originate from mRNA, cloned V genes or genomic templates. The donor V PCR DNA is denatured and annealed to the M13 cassette containing the recipient V to be supplanted. The second strand is synthesized, transfected into bacteria and mutant plaques selected by hybridization. Since restriction sites in primers are not required, altered primer-encoded amino acids are avoided. Further, the PCR donor piece can be of any length if it shares homology with the recipient gene. This allows construction and expression of complete gene replacements and chimeras. This method is also applicable to V "humanization" and studying sets of homologous genes containing polymorphic or evolutionary disparities. The potential uses of the technique are discussed.     

A selenocysteine-containing selenium-transport protein in rat plasma

A selenocysteine-containing rat plasma protein (selenoprotein P) was examined for a possible role in the transport of selenium in the rat. A time-course study of the localization of injected 75Se from [75Se]selenite indicated that one-half of the selenium was sequestered by liver tissue 1 h after injection and that one-fourth of the 75Se in the plasma was attached to selenoprotein P 3 h after injections. By 25 h there was little 75Se in plasma, and much of the 75Se had accumulated in nonhepatic tissues. 75Se was incorporated into selenoprotein P by liver slices in a process that was sensitive to the protein synthesis inhibitor cycloheximide. The fate of 75Se from intracardially injected 75Se-labeled selenoprotein P was followed in rats maintained on selenium-deficient and selenium-sufficient diets. Substantially more of the injected 75Se was present per gram wet weight in the testes and kidneys than the livers of the selenium-deprived rats after 5 h. The results indicate that selenoprotein P is synthesized in rat liver and that it transfers selenium from the liver to extrahepatic tissues.     

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model

The MHC class I allochimeric protein containing donor‐type epitopes on recipient‐type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [α1h1/u]‐RT1.Aa that contains donor‐type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient‐type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL‐1β a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients. genesis 48:8–19, 2010. © 2009 Wiley‐Liss, Inc.     

Relationships between cardiac hyperactivity, oxygen tension, and release of vasodilator material in coronary autoregulation of guinea-pig hearts

Increases in cardiac activity induce autoregulatory coronary vasodilation. The intermediate steps which trigger this process are thought to be myocardial hypoxia which induces the release of vasodilator mediator(s). The present study examines the relationships between mechanical activity, oxygen tension, and release of vasodilator material in isolated perfused hearts. Guinea-pig isolated hearts were perfused in series, the effluent from donor hearts being regassed prior to entry to recipient hearts. Histamine (1 microgram) and isoproterenol (10 ng) increased the rate and tension of donor hearts and produced predominant coronary vasodilator responses which were followed by the appearance of vasodilator material in the recipient (falls in perfusion pressure, 9.8 +/- 1.1 and 9.1 +/- 2.5 mmHg) (1 mmHg = 133.322 Pa). Exposure of donor hearts to hypoxia also caused vasodilatation and release of vasodilator material (fall in pressure, 11.4 +/- 1.6 mmHg). Pacing-induced tachycardia (6 Hz) of donor hearts promoted the release of vasodilator material, the fall in recipient heart pressure being 11.5 +/- 1.8 mmHg. This was abolished by beta-adrenoceptor blockade and when donor hearts were from reserpine-pretreated guinea pigs. In was concluded that pacing released endogenous catecholamines which in turn released the vasodilator material. Pacing per se did not cause vasodilatation or release of the vasodilator. The Po2 of perfusates from donor hearts was reduced by pacing at 5 Hz (25.7 +/- 5.2 mmHg) and by isoproterenol (10 ng, 32.0 +/- 3.7 mmHg), indicative of an elevated oxygen extraction. The isoproterenol-induced falls in Po2 were abolished by beta-adrenoceptor blockade. However, the pacing-induced falls in Po2 persisted, the values occurring before (25.7 +/- 5.2 mmHg) and after propranolol (45.7 +/- 4.5 mmHg) and before (32.1 +/- 1.1 mmHg) and after practolol (27.3 +/- 4.1 mmHg) not differing significantly (p greater than 0.05). These falls in perfusate Po2 were not accompanied by coronary vasodilatation or release of vasoactive material. Perfusate Po2 changes could therefore be dissociated from the coronary vasodilatation and vasoactive material release, suggesting that hypoxia may not be a prerequisite for the metabolic autoregulatory vasodilatation in response to myocardial hyperactivity induced by cardiac stimulants.     

Cardiac injury protection from mouse bone marrow stromal cells with in utero transplantation followed by secondary postnatal boost

Limited donor-cell engraftment to the injured tissue restricts therapeutic efficacy of stem cell transplantation. Herein, we proposed an alternative strategy by using in utero transplantation (IUT) to create mixed-chimerism environment in recipients and to facilitate donor-cell engraftment followed by postnatal secondary boost with the same cells. Mouse bone marrow stromal cells (BMSCs) were used as the xenogenic donor cells and given into rat fetus as an early exposure of IUT treatment. The engraftment potential was analyzed for the presence of BMSCs by flow cytometry or PCR in recipient tissues. The function of a second boost of mouse BMSCs, in terms of cardioprotection, was tested by given 1×10? cells to rat IUT hearts with ischemia/reperfusion (IR) injury that was induced by a 45 min of left coronary ligation and released for 72 h. Mouse BMSCs demonstrated an immunosuppressive effect when mixed with mouse or rat lymphocytes. IUT treatment only caused few BMSCs engrafted to fetal (embryonic day 20) and adult (4 weeks after birth) rat organs including heart, but engraftment was increased in hearts of the IUT rats after second boost. This was coincided with attenuation of cardiac injury caused by IR. Interestingly, an up-regulation of CXC chemokine receptor type 4 (CXCR4) was seen when BMSCs were exposed to hypoxia. This indicates that enhanced engraftment of mouse BMSCs to post-ischemic rat hearts possibly is dependent on CXCR4. Moreover, results of flow cytometry demonstrated that the presence of CD34? cells in rat IUT hearts with IR injury was increased. These observations suggest that enhanced engraftment of donor BMSCs to rat IR hearts by CXCR4 may recruit endogenous CD34? cells of recipients which in turn protects heart against IR. This also supports the notion of fetal preconditioning with BMSC enhances the efficiency of progenitor cell-mediated organ protection after a postnatal second boost in xeno-transplantation.     

Acetaldehyde and cardiac protein synthesis in the rat in vivo

Protein synthesis was measured in the hearts of rats exposed to acetaldehyde vapour for 21 days. Exposure to acetaldehyde significantly increased heart weight (expressed as % body weight) but was without effect on the rate of synthesis of mixed cardiac proteins. Concentrations of RNA in the hearts were not altered by acetaldehyde exposure, indicating no change in RNA activity for protein synthesis.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Noncoordinate synthesis of the vitellogenin proteins in tissues of Drosophila grimshawi

In analyzing the in vitro pattern of protein synthesis by the fat body and ovaries of the Hawaiian species Drosophila grimshawi, we have found that the ovaries synthesize much more protein than the female fat body and that the majority of the synthesized proteins are retained by the ovarian tissues. In contrast, the fat body secrets most of the proteins into the culture medium. Vitellogenins are the major class of proteins synthesized and released into the medium by both tissues. The synthesis of the three vitellogenin proteins (V1, V2, V3) is noncoordinate in the two tissues. Ovaries synthesize much more of the V2 protein, less V1 and very little V3, whereas fat body synthesizes more V1 protein with lesser quantities of the other two. The follicle cells were identified as the site of ovarian vitellogenin synthesis in D. grimshawi, confirming the findings in D. melanogaster. In D. grimshawi, the three vitellogenins are synthesized by the follicle cells in a noncoordinate and developmentally regulated manner. V2 and V1 are the predominant proteins at the onset of vitellogenesis (S8-9); their production peaks together with that of V3 a few hours later (S10) and then decreases to quantities equal to that of V3 during early choriogenesis (S11). During active choriogenesis (S12), V2 and V1 cease to be synthesized, but V3 synthesis continues. The vitellogenins synthesized by the follicles in vitro are released into the medium and not incorporated into the oocyte.