In vivo phosphorylation of isocitrate lyase inEscherichia coli andAcinetobacter calcoaceticus

Isocitrate lyase inEscherichia coli and inAcinetobacter calcoaceticus is phosphorylated when the cells are grown with acetate as the sole carbon source in low-phosphate mineral salts medium containing³²P inorganic phosphate. The level of³²P incorporation into the enzyme in both microorganisms appears to be constant throughout the entire growth cycle. Further, theresults of immunoblots and rocket immunoelectrophoresis suggest that the amount of isocitrate lyase protein, although at different levels in each microorganism, also remains constant throughout the growth cycle.     

Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.     

Hypobaric hypoxia (380 Torr) decreases intracellular and total body water in goats

The effects of prolonged hypoxia on body water distribution was studied in four unanesthetized adult goats (Capra lircus) at sea level and after 16 days in a hypobaric chamber [(380 Torr, 5,500 m, 24 +/- 1 degrees C); arterial PO2 = 27 +/- 2 (SE) Torr]. Total body water (TBW), extracellular fluid volume (ECF), and plasma volume (PV) were determined with 3H2O, [14C]inulin, and indocyanine green dye, respectively. Blood volume (BV) [BV = 100PV/(100 - hematocrit)], erythrocyte volume (RCV) (RCV = BV - PV), and intracellular fluid (ICF) (ICF = TBW - ECF) and interstitial fluid (ISF) (ISF = ECF - PV) volumes were calculated. Hypoxia resulted in increased pulmonary ventilation and arterial pH and decreased arterial PCO2 and PO2 (P less than 0.05). In addition, body mass (-7.1%), TBW (-9.1%), and ICF volume (-14.4%) all decreased, whereas ECF (+11.7%) and ISF (+27.7%) volumes increased (P less than 0.05). The decrease in TBW accounted for 89% of the loss of body mass. Although PV decreased significantly (-15.3%), BV was unchanged because of an offsetting increase in RCV (+39.5%; P less than 0.05). We conclude that, in adult goats, prolonged hypobaric hypoxia results in decreases in TBW volume, ICF volume, and PV, with concomitant increases in ECF and ISF volumes.     

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium

In a variety of lung diseases the respiratory epithelial surface must contend with an increased burden of neutrophil elastase (NE). One candidate for augmenting epithelial anti-NE protection is the secretory leukoprotease inhibitor (SLPI). In vitro evaluation demonstrated that 96 +/- 1% of the recombinant SLPI (rSLPI) molecules were capable of inhibiting NE, with an association rate constant of 7.1 +/- 0.1 X 10(6) M-1.s-1. Evaluation of rSLPI after in vitro and in vivo aerosolization showed that aerosolization did not alter rSLPI. Aerosolization of a single dose of 50 mg rSLPI to sheep resulted in a fourfold increase of the anti-NE capacity in epithelial lining fluid (ELF) at 3 h, with a half-life in ELF of 12 h. After aerosolization some rSLPI appeared in lung lymph. Simultaneous aerosolization of rSLPI and recombinant alpha 1-antitrypsin (rAAT) demonstrated a molar ratio of the concentration in lymph to the concentration in ELF 3 h after the aerosol eightfold higher for rAAT than for rSLPI. Overall, these observations demonstrate that it is feasible to use aerosolized rSLPI to directly augment the anti-NE capacity of the lung, particularly on the pulmonary epithelial surface.     

Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Ontogeny of endocrine cells in the respiratory system of Syrian golden hamsters

The ontogeny of protein gene product 9.5 (PGP 9.5), serotonin (5–HT), calcitonin gene-related peptide (CGRP), and calcitonin (CT) immunoreactivity was evaluated in small-granule endocrine cells of hamster laryngotracheal epithelium from fetal day 11 to adulthood. Two centrifugal (proximal-to-distal) patterns of differentiation occur. The first pattern begins during fetal life. Endocrine cells, single and clustered in groups (presumptive- or protoneuroepithelial bodies, pNEBs), initially co-localize immunostaining for PGP 9.5, 5–HT, and CGRP in the larynx and proximal 2/3 of the trachea on day 12 and spread to the caudal trachea on day 13. 5–HT disappears fleetingly during the 24 h preceding birth; otherwise immunoreactivity for all three substances persists into adulthood. The clusters of endocrine cells survive beyond birth but are so diluted by expansion of the nonendocrine epithelium as to become inconspicuous. Since innervation was not actually observed, these clusters may persist as pNEBs, without developing connections to afferent or efferent nerve fibers. The second pattern concerns single small-granule cells stainable for CGRP but not for 5–HT. These cells first appear in the larynx and cartilaginous part of the cranial trachea on postnatal day 3, and in the middle and caudal trachea, on day 5. The cells increase in number on day 7. In adults, they predominate among endocrine cells of the cartilaginous region. A subset of these cells begins to co-express CT proximally on postnatal day 10, reaching the caudal end of the trachea by 3 weeks. A few elements of the older 5–HT-positive population may also become immunoreactive for CT in juvenile hamsters.