In organello formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins

The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected approximately 20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2Delta mutation increases the petite-inducing phenotype of an abf2Delta mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of rho(+) mtDNA.     

Outcome following cardiopulmonary resuscitation in the neonate requiring ventilatory assistance

BACKGROUND: there is limited data regarding the clinical characteristics and outcome of the neonate requiring ventilatory assistance who develops persistent bradycardia (PB) requiring cardiopulmonary resuscitation (CPR). OBJECTIVES: (1) to determine the percentage of newborn infants requiring respiratory assistance who develop PB and require CPR as part of resuscitation; (2) the associated clinical events; and (3) the short term outcome. METHODS: the medical charts of infants admitted to a neonatal intensive care unit who developed PB, defined as a heart rate <80 beats/min requiring CPR, were retrospectively reviewed. RESULTS: for 3 years, 39 (2.6%) of 1485 infants exhibited 62 episodes of PB requiring CPR; this represents 5.6% of 695 intubated infants. Fourteen (36%) infants rapidly responded to chest compressions only with restoration of heart rate within 2 min; termed brief CPR. None died in-hospital. Twenty-five (64%) infants required prolonged chest compressions, i.e. >2 min (termed prolonged CPR); 21 also received epinephrine. The median postnatal age at onset of CPR was 20 days (range 1-148 days) and the duration of CPR was 10 min (range 3-73 min). The more common medical conditions that may have contributed to the PB included severe bronchospasm associated with chronic lung disease (CLD) (n=6), shock associated with sepsis (n=4) and necrotizing enterocolitis (NEC) (n=2), pneumothorax (n=2), inadequate or improper ventilation (n=3), other (n=8). Nineteen (76%) infants died: 13 within 24 h of the event and six from 3 to 194 days following CPR. At 18 months follow-up, four of the six infants evaluated have a moderate to severe neurodevelopmental deficit. Of the nine infants requiring brief CPR who were evaluated, five are developing normally and four have a moderate to severe neurodevelopmental deficit. CONCLUSION: CPR in the neonate who requires ventilatory assistance is not uncommon. When brief in nature, mortality is low and short-term outcome is likely to be determined by the underlying medical condition. When CPR is prolonged, mortality is high and short-term outcome is poor.     

Negative regulation of angiotensinogen gene expression by glucocorticoids in fetal sheep liver

The effect of glucocorticoids in regulating liver angiotensinogen gene expression was studied in chronically instrumented fetal sheep during the last trimester of gestation and was compared with the expression of other hepatic genes (prothrombin, factor IX, and albumin). Four sets of twins were studied at 118 d of gestation, and three sets were studied at 138 d of gestation (term, 145 d). One of each set of twins was infused intraperitoneally with cortisol (5 mumol.mL-1.h-1) for 48 h, whereas the other twin received the same volume (1 mL/h) of normal saline. Plasma cortisol concentration increased from 0.32 +/- 0.12 and 2.7 +/- 0.12 nmol/100 mL to 44.2 +/- 20.0 and 37.7 +/- 8.2 nmol/100 mL in 118- and 138-d fetuses, respectively, during the cortisol infusion; no changes were observed in fetuses infused with saline alone. At the end of the infusion period, the animals were anesthetized, the fetal liver was removed, and total cellular RNA was isolated and probed for angiotensinogen, prothrombin, factor IX, and albumin. The results demonstrated that cortisol infusion decreased angiotensinogen mRNA by 61% in 138-d fetuses and albumin mRNA expression by 2.4-fold in 118-d fetuses and by 3.4-fold in 138-d fetuses. On the other hand, cortisol had no effect on fetal factor IX gene expression but increased prothrombin mRNA levels by 65% in 118-d fetuses and 62% in 138-d fetuses. Taken together, our results suggest that, during fetal life, angiotensinogen gene expression is negatively regulated by glucocorticoids. This effect is not universal because cortisol increases fetal prothrombin gene expression.     

Movement disorder of premature infants with severe bronchopulmonary dysplasia: a new syndrome

A previously unrecognized, striking movement disorder has been observed in 10 premature infants with severe bronchopulmonary dysplasia. Chronic hypoxemia, hypercarbia, bronchospasm, and inadequate nutrition were present in all. The movement disorder developed from approximately the third postnatal month. The dominant movements involve the limbs, neck, trunk, and oral-buccal-lingual structures. The limb movements were most prominent distally and consisted of rapid, random, jerky movements (similar to chorea) and "restless" movements (similar to akathisia). Similar movements of the neck and face were observed; tongue movements had a "darting" quality. The oral-buccal-lingual movements were similar to the dyskinesia of older patients. Movements were exacerbated during episodes of respiratory failure and attenuated during sleep. All infants exhibited feeding disorders, largely due to tongue movements. In 3 infants treated with clonazepam, there was striking improvement in motor function, including feeding. The natural history was partial or complete resolution or a static course. Thus, of the 7 surviving infants, the movements were absent (without therapy) at 15, 18, and 30 months of age. In the remaining 4 infants (3 of whom receive clonazepam), the movements, though attenuated, persisted at 6, 12, 15 and 21 months of age, respectively. Neuropathologically, 1 infant showed neuronal loss with astrocytosis in caudate, putamen, globus pallidus, and thalamus. These data defined a previously unrecognized extrapyramidal movement disorder of infants with severe bronchopulmonary dysplasia; pathogenesis may be related to chronic hypoxemia.     

Developmental regulation of angiotensinogen gene expression in sheep

It has been suggested that the liver is not the main source of angiotensinogen during fetal life in rats, but that the kidney is an important site of fetal angiotensinogen synthesis. In an effort to determine if this phenomenon is specific to the rat or applicable to other species, we compared the ontogenic changes in hepatic and renal angiotensinogen mRNA expression in fetal (60, 90, 118, and 138 d of gestation, term being 145 d), newborn (7 d postnatal), and adult sheep. Total RNA was extracted, subjected to Northern blotting and hybridized using a full-length rat radiolabeled antisense RNA. Angiotensinogen mRNA sequences were detected in all fetal liver samples and appeared to increase 3-fold from 60 to 138 d gestation and then to decrease after birth. In contrast, angiotensinogen mRNA could not be detected in renal cortical tissue of 118 or 138 d fetuses, or newborn or adult sheep. We conclude that, unlike in the rat, liver angiotensinogen gene expression is detectable during the 2nd trimester of gestation in sheep and is developmentally regulated. Furthermore, in contrast to the fetal rat, angiotensinogen mRNA sequences were undetectable in fetal sheep kidney.     

Is chloride depletion an important contributing cause of death in infants with bronchopulmonary dysplasia?

A retrospective analysis of infants with bronchopulmonary dysplasia requiring prolonged hospitalization (greater than 100 days) was carried out to determine those factors associated with fatal outcome. Twenty-three infants made up the study population. Eleven infants died and 12 survived (survivors). No differences were noted between the groups regarding ventilator requirement, radiographic changes, and medication use (digoxin, aldactazide), except for furosemide which was used twice as frequently in the group of infants who died v the group of infants who survived (P less than .001). Differences noted between the groups included moderate hypochloremia (chloride less than 80 mEq/L) in all 11 infants who died v six of 12 survivors, severe hypochloremia (chloride less than 70 mEq/L) in the nine of 11 infants who died v two of 12 survivors, metabolic alkalosis (pH greater than 7.45) in nine of 11 infants who died v three of 12 survivors, hypertension (systolic BP greater than 113 mm Hg) in eight of 11 infants who died v one of 12 survivors, decrease in head growth in ten of the 11 infants who died v one of the 12 survivors; these differences were all significant (P less than .001). The metabolic alkalosis and head growth changes appear to be related to the hypochloremia. The data suggest that chloride deficiency may be an important contributing factor in the genesis of poor outcome in infants with bronchopulmonary dysplasia and that close attention to chloride supplementation might influence outcome.