Conversion of phosphatidylglycerol to lyso(bis)phosphatidic acid by alveolar macrophages

We report here studies of the synthesis of lyso(bis)phosphatidic acid [L(b)PA] by normal and BCG-elicited rabbit alveolar macrophages. This study was prompted by our earlier observations that 1) alveolar macrophages did not synthesize L(b)PA de novo despite its abundance in these cells, 2) BCG-elicited cells contained only one-quarter the amount of L(b)PA as normal cells, and 3) the turnover of arachidonate in L(b)PA led to hydroxyeicosatetraenoic acid and leukotriene synthesis. We found that exogenous phosphatidylglycerol (PG) was specifically converted to L(b)PA by both types of cells although BCG-elicited cells had only one-quarter the synthetic capacity of normal cells. Other phospholipids were found to become cell associated but were not significantly metabolized. Both glycerol moieties and the phosphate were incorporated into the product L(b)PA. However, substitution of the ester with an alkyl linkage in position 1 blocked the conversion of PG to L(b)PA. Most of the alkylphosphatidylglycerol was converted to phosphatidylcholine and phosphatidylethanolamine. This result implied that catabolism of the acyl group in position 1 was essential for L(b)PA synthesis. Because alveolar macrophages are present in a surfactant-rich milieu, we suggest that surfactant provides a source of PG for macrophage synthesis of L(b)PA in situ. It is interesting that the surfactants from rabbits challenged with BCG have a significant decrease in PG content.     

Right ventricular function in the hypoxaemic fetal sheep

Right ventricular function was investigated in seven fetal sheep (125-130 days gestation) hypoxaemic at a mean of 5 days postoperation, and were compared to nine normoxaemic fetal sheep of the same gestation. Arterial O2 and CO2 tensions, pH, and haematocrit values for the hypoxaemic and normoxaemic fetuses were 15.6 +/- 1.0 vs. 20.6 +/- 1.8 torr, 49.4 +/- 4.1 vs. 46.1 +/- 1.6 torr, 7.38 +/- 0.02 vs. 7.39 +/- 0.02, and 29 +/- 7.5 vs. 31 +/- 5.3%, respectively. Right ventricular output and stroke volume were similar in the two groups, 241 +/- 57 vs. 247 +/- 75 ml X min-1 X kg-1 and 1.5 +/- 0.4 vs. 1.5 +/- 0.4 ml X kg-1, respectively. Filling and afterload pressures were also similar in the hypoxaemic and normoxaemic fetuses with right atrial pressure of 3.0 +/- 1.0 vs. 3.7 +/- 1.2 mmHg, and arterial pressure of 42 +/- 5 vs. 43 +/- 4 mmHg, respectively. Ventricular function curves were produced by rapid withdrawal and re-infusion of fetal blood producing curves with a steep ascending limb and a plateau phase. The breakpoint joining the limbs of the control function curve for the hypoxaemic and normoxaemic fetuses were right atrial pressure 2.9 +/- 1.0 vs. 3.4 +/- 1.2 mmHg and a stroke volume of 1.5 +/- 0.5 vs. 1.5 +/- 0.4 ml X kg-1, respectively. Linear regression of stroke volume against arterial pressure from 30-90 mmHg during infusions of nitroprusside and phenylephrine at right atrial filling pressures greater than breakpoint was stroke volume = 0.018 ml X kg-1 X mmHg-1 arterial pressure +/- 2.25 ml X kg-1. This equation is not different from that calculated in normoxaemic fetuses, and demonstrates that the fetal right ventricle is quite sensitive to changes in arterial pressure. These data indicate that reduction in fetal oxygen content by an estimated 40% does not affect fetal right ventricular function.     

Dietary-induced increase in lactase activity and in immunoreactive lactase in adult rat jejunum.

Adult rats that had been fed on a low-starch high-fat diet for 7 days were force-fed with either the same diet or isoenergetic diets containing 40% of energy as either sucrose or lactose. Within 12h, the increase in jejunal lactase activity in sucrose- and lactose-fed rats was accompanied by a corresponding increase in immunoreactive lactase protein.     

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Uridine 5′-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5′-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5′-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Classification and nomenclature of all human homeobox genes

Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.     

Is the nectar redox cycle a floral defense against microbial attack?

Many angiosperms use a remarkable reproductive strategy that relies on attracting animals (insect, avian or mammalian pollinators) to transfer pollen between plants. Relying on other organisms for sexual reproduction seems evolutionarily untenable, but the great diversity of angiosperms illustrates how highly successful this strategy is. To attract pollinators, plants offer a variety of rewards. Perhaps the primary floral reward is floral nectar. Plant nectar has long been considered a simple sugar solution but recent work has demonstrated that nectar is a complex biological fluid containing significant and important biochemistry with the potential function of inhibiting microbial growth. These results lead the way to novel insights into the mechanisms of floral defense and the co-evolution of angiosperms and their pollinators.     

Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes

Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro.     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart.

The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.