Morbidity and mortality associated with rewarming hypothermic very low birth weight infants.

OBJECTIVES: In very low birthweight (VLBW) infants, hypothermia is associated with poor outcomes. The goal of this study is to assess the relationship between the rate of rewarming these babies and their outcomes. METHODS: This is a retrospective cohort study of 98 inborn VLBW infants who were hypothermic (<36°C rectally) upon admission to the NICU. A logistic regression model was used to examine the relationship between the rates of rewarming and time to achieve euthermia and the following outcomes: death, intraventricular hemorrhage, severe intraventricular hemorrhage, bronchopulmonary dysplasia, necrotizing enterocolitis and retinopathy of prematurity. RESULTS: Prolonged rewarming time was associate with increased odds of mortality (OR 1.273 95% CI 1.032-1.571). No associations between rewarming rates and any of the outcomes were seen. Once birthweight was included in a multiple logistic regression model, the association between mortality and rewarming time was no longer significant. Outcomes that were not associated with either rate or time of rewarming (even in a univariate model) were: bronchopulmonary dysplasia, intraventricular hemorrhage, severe intraventricular hemorrhage, necrotizing enterocolitis and retinopathy of prematurity. CONCLUSION: In moderately hypothermic VLBW infants, after accounting for birthweight, no association between rewarming and outcome is seen.

What is the rationale for the use of granulocyte and granulocyte-macrophage colony-stimulating factors in the neonatal intensive care unit?

UNLABELLED: Neonatal sepsis remains an unsolved major contributor to morbidity and mortality. In the 1980s the promise of augmenting immune function using pooled intravenous gammaglobulin to supplement the exceedingly low levels of immunoglobulin G in premature infants failed to demonstrate a clear advantage. Similarly, cytokine augmentation of cellular function in the 1990s largely appeared to be suffering the same fate. However, both results may arise from a problem in experimental design where the combination of both treatments may be necessary along with specific antibody. For example, in vitro, independently of an array of other humoral and cellular immature immune system issues, opsonization of bacteria is improved in the presence of antibody. The question is whether the same result can be achieved in vivo. No experiments have been reported that directly test this hypothesis. CONCLUSION: More investigation is needed in this challenging area of neonatal research.

Effects of prenatal steroids on water and sodium homeostasis in extremely low birth weight neonates.

OBJECTIVE: We sought to determine if prenatal steroid (PNS) treatment affects water and sodium (Na) balance in extremely low birth weight infants (<1000 g). METHODS: PNS treatment enhances lung maturation in preterm infants and induces maturation of renal tubular function and adenylate cyclase activity in animals. We compared water and Na homeostasis for the first week of life in those infants whose mothers received steroids before delivery (PNS: n = 16) to those who did not (nonsteroid group [NSG]: n = 14). The data were collected prospectively, but PNS treatment was not given in a randomized manner. Fluids were initiated at 100 to 125 mL/kg/d and adjusted every 8 to 12 hours to allow a daily weight loss of 150 mmol/L compared with 36% of the NSG infants. PNS infants had a higher cumulative Na excretion at day 2 of life (10 +/- 2 mmol/kg vs 6 +/- 1 mmol/kg) but a less negative cumulative Na balance at 1 week (-10 mmol/kg vs -14 mmol/kg). CONCLUSION: PNS treatment was associated with lower estimated insensible water loss, a decreased incidence of hypernatremia, and an earlier diuresis and natriuresis in extremely low birth weight neonates. We speculate that PNS effects these changes through enhancement of epithelial cell maturation improving skin barrier function. PNS treatment may also enhance lung Na, K-ATPase activity leading to an earlier postnatal reabsorption of fetal lung fluid increasing extracellular volume expansion to help prevent hypernatremia.

Preliminary report: rhG-CSF may reduce the incidence of neonatal sepsis in prolonged preeclampsia-associated neutropenia.

OBJECTIVES: To determine whether adjunctive therapy with recombinant human granulocyte colony-stimulating factor (rhG-CSF) could reverse the neutropenia and reduce the incidence of sepsis (/=3 consecutive days in the first postnatal week) preeclampsia-associated neutropenia (absolute neutrophil count [ANC] <1500/mm3). Neutrophilic responses and the incidence of neonatal sepsis in the next 28 postnatal days were compared with 13 case-matched control neonates who also had prolonged preeclampsia-associated neutropenia. Sepsis was defined as at least one positive blood culture in a newly symptomatic neonate treated with antibiotics for >/=7 days. RESULTS: No significant differences existed among the three groups in the birth weight, gestational age, sex, growth retardation, method of delivery, magnitude of respiratory support, use of surfactant, usage of intravascular catheters, or in the initial (pretreatment) ANC. The average baseline ANC (pretreatment) in the 10 microgram rhG-CSF group was 815 +/- 169/mm3 (mean +/- SEM), in the 5 microgram group it was 786 +/- 165/mm3, and in the conventional group it was 965 +/- 283. Eighteen of 28 (64%) neonates with preeclampsia-associated neutropenia were neutropenic at birth, the other 10 (36%) had normal neutrophil counts at birth but subsequently developed >/=3 days of neutropenia between 24 and 120 hours after birth. The ANC increased by 2-fold at 24 hours, by 4-fold at 72 hours, and 14-fold by the 7th day in the 10-microgram group. In the 5-microgram group, a 2-fold and 5-fold increase occurred at 72 hours and 7 days, respectively. In the conventionally-treated group, only a 4-fold increase was seen as late as 7 days after achieving entry criteria. Sepsis was observed in 13% (2/15) of the rhG-CSF-treated neonates compared with an incidence of 54% (7/13) in the conventionally-treated neonates. Conclusions. rhG-CSF increases the ANC significantly (at 10 microgram/kg/day x 3 days) and reduces the incidence of neonatal sepsis in critically ill ventilated neonates with prolonged preeclampsia-associated neutropenia when compared with conventional therapy. A future prospective, randomized, and blinded trial is needed to validate the beneficial effects of prophylactic rhG-CSF therapy in neonates with prolonged preeclampsia-associated neutropenia.

Human granulocyte colony-stimulating factor may improve outcome attributable to neonatal sepsis complicated by neutropenia.

OBJECTIVES: To determine whether adjunctive therapy with recombinant human granulocyte colony-stimulating factor (rhG-CSF) could reverse sepsis-associated neonatal neutropenia and improve neonatal survival compared with conventional therapy in a phase I/II-type trial. STUDY DESIGN: An intravenous infusion of rhG-CSF (10 microg/kg/d x 3 d) was administered to 14 septic neutropenic neonates. Neutrophilic responses and outcome of these neonates were compared with 11 concurrently treated, retrospectively selected, case-matched control septic patients identified by using a search of medical records coded for sepsis with neutropenia (>/=24 hours). RESULTS: Seven neonates with early-onset sepsis with neutropenia at birth and seven neonates with late-onset sepsis plus neutropenia (all with necrotizing enterocolitis) were entered in the rhG-CSF treatment group. Results were compared with a conventional therapy control group (five early onset, six late onset). No significant differences existed in the birth weight, gestational age, use of antibiotic therapy, magnitude of respiratory support, severity of metabolic acidosis, use of vasopressors, or other supportive therapy between the two groups. In the rhG-CSF-treated group and in the conventionally treated control group, the absolute neutrophil count (ANC) (mean +/- SEM) was 585 +/- 138 and 438 +/- 152, respectively. The ANC increased to more than baseline in the rhG-CSF-treated group by 10-fold versus 2-fold at 24 hours, 18-fold versus 4-fold at 48 hours, 24-fold versus 5-fold at 72 hours (significant by one-way analysis of variance in the rhG-CSF group only), and 29-fold versus 16-fold at 7 to 10 days when compared with the conventional therapy group. There were no nonresponders in the rhG-CSF group by 24 hours after the first dose of study drug. Monocyte cell counts also increased significantly in both groups by 7 days after entry into this protocol but remained within normal range for age. No clinically significant effect on lymphocytes, erythrocytes, or platelet counts was noted. Thirteen patients in the rhG-CSF-treated group (92%; 13 out of 14) and five in the conventionally treated group (55%; 5 out of 11) survived to 28 days after the onset of the signs of sepsis. No adverse effects were noted in the rhG-CSF-treated group. CONCLUSIONS: rhG-CSF can increase the neutrophil count in critically ill septic neutropenic neonates. This finding suggests that rhG-CSF may be effective in a therapeutically useful time frame to treat septic neonates with neonatal neutropenia attributable to bone marrow suppression or neutrophil consumption. Future randomized trials are needed to validate the beneficial effects of rhG-CSF and to determine whether any significant side effects of therapy exist.

Effects of increased red cell mass on subclinical tissue acidosis in hyaline membrane disease.

AIM: To determine whether there are subclinical deficits in oxygen delivery in ventilated premature neonates. METHOD: Ventilated premature neonates weighing less than 1500 g, who were transfused for anaemia or who were given colloids for clotting abnormalities (or oedema), were haemodynamically monitored during the first week of life. Calf muscle surface pH (pH) was measured in conjunction with peripheral limb blood flow by occlusion plethysmography. RESULTS: Packed red blood cell transfusions corrected a subclinical regional tissue acidosis (low tpH) without affecting arterial pH or limb blood flow. This observation also correlated with an increase in regional oxygen delivery. The data were also suggestive of a pattern of pathological, supply dependent, oxygen delivery and are similar to other observations made in adults with adult respiratory distress syndrome. CONCLUSIONS: Packed red blood cells increase regional oxygen delivery and tissue surface pH. In contrast, colloid infusion provided no substantial cardiovascular or metabolic benefit to these patients and should be avoided when oxygen delivery is at issue and when there may be leaky pulmonary capillaries.

Management of 168 neonates weighing more than 2000 g receiving intrapartum chemoprophylaxis for chorioamnionitis. Evaluation of an early discharge strategy.

OBJECTIVE: To determine whether sequential laboratory and clinical evaluations during the first 3 days of postnatal life can be used to safely limit the duration of antibiotic therapy for term neonates whose mothers received intrapartum antibiotic treatment for intra-amniotic infection (ie, chorioamnionitis). METHODS: Since postpartum neonatal body fluid cultures can be falsely negative because of transplacental passage of maternal antibiotics, we prospectively followed up 6620 pregnancies for 28 months (December 1991 through March 1994) for the occurrence and treatment of chorioamnionitis. Neonatal antibiotic therapy was initiated and limited to 3 days or continued for 7 days or more in neonates with abnormal laboratory values or clinical signs that were consistent with sepsis on day 3 of postnatal age. Both groups were observed in the hospital for 24 to 48 hours after antibiotics were discontinued. RESULTS: Of the 6620 pregnancies, 158 infants (2.4%) born to 155 mothers received intrapartum antibiotics for chorioamnionitis; 10 additional neonates diagnosed as having chorioamnionitis were transported from other hospitals (N = 168). Because of the absence of signs and negative cultures, 82% (137/168) were treated with antibiotics for 3 days, while 18% (31/168) received 7 days or more of therapy. In 84% of the 3-day group, discharge was accomplished by postnatal day 4 or 5, whereas all of the 7-day or more group were discharged after day 8. Follow-up calls placed 1 month after discharge disclosed no adverse outcomes or hospital readmissions in any of the infants in this survey. CONCLUSIONS: Neonates with infection who are born to mothers pretreated with antibiotics for intra-amniotic infection can be reliably identified less than 72 hours after birth and treated appropriately. As 82% of at-risk patients are asymptomatic and have a negative body fluid culture, our data support the position that a full course of antibiotic therapy can be restricted to only those patients with clinical or laboratory signs of sepsis (18%). This will effective reduce the average length of hospital stay for intrapartum-treated neonates by a minimum of 3 to 4 days compared with a commonly used empiric therapy approach of continuing medication for 7 days or more. Perhaps hospital discharge can be further shortened if a 1- to 2-day posttreatment observation period is eliminated for all patients except those with a positive body fluid culture.

Recombinant human interleukin-3 enhances the in vitro survival of mononuclear phagocytes from the peripheral blood of very low birth weight premature infants at birth.

Interleukin 3 (IL-3) is a pluripotent hematopoietic growth factor that stimulates proliferation, differentiation, and function of multiple cell lineages. We examined the effects of recombinant human IL-3 (rhIL-3) on peripheral blood mononuclear cells (MNC) isolated from 18 premature human newborns with birth weights between 600 and 1,500 g at birth and at 2 and 4 weeks of age, from 7 full-term neonates, and from 26 normal adult volunteers. After 2 weeks in liquid culture, rhIL-3 treatment was associated with a six- to nine-fold increase in the survival of MNC from very low birth weight (VLBW) neonates. In the absence of rhIL-3, VLBW neonatal MNC exhibited a low survival rate. MNC from 6 of 7 full-term neonates responded similarly to rhIL-3 with an eight-fold increase in survival. In contrast, rhIL-3 showed only a 20-30% increase in the survival of adult MNC (p = NS). When analyzed by immunofluorescent microscopy using monoclonal antibodies to phenotypic markers characteristic of individual MNC lineages, 70-80% of the surviving VLBW neonatal MNC were mononuclear phagocytes, while 70-80% of the surviving adult MNC were T cells. Full-term MNC cultures displayed a population of cells with an intermediate phenotype. Following rhIL-3 treatment, surviving MNC from term neonates displayed 35% T cells and 53% mononuclear phagocytes which was not significantly different from untreated MNC. rhIL-3 treatment was associated with a seven- to twelve-fold increase in the number of progenitor cells (CD34+) from VLBW neonatal blood and a three- to five-fold increase in the number of adult progenitor cells. Full-term neonates had a lower percentage of CD34+ cells than VLBW neonates, and this was not significantly altered by the rhIL-3 treatment. We conclude that rhIL-3 increases the number of mononuclear phagocytes that survive in culture following isolation from the peripheral blood of VLBW neonates. These in vitro studies may have a predictive value for in vivo studies utilizing combinations of hematopoietic growth factors to enhance neonatal host defense.

Nutritional management and the multisystem organ failure/systemic inflammatory response syndrome in critically ill preterm neonates.

Current information and concepts regarding unique features and practical aspects of metabolism and the nutritional management of critically ill, very low birthweight neonates are reviewed in this article. The use of "gut priming" (early hypocaloric minimal enteral feeding) and parenteral nutrition and their application to the treatment of specific disease states is discussed. The concepts of critical oxygen delivery and multiorgan failure and their impact on nutritional management in adults show striking similarities to metabolic observations of progressively deteriorating, sick neonates.

Effect of granulocyte colony-stimulating factor on preeclampsia-associated neonatal neutropenia.

Absolute neutropenia lasting longer than 72 hours after birth occurred in four very low birth weight neonates with a maternal history of severe pregnancy-induced hypertension, and was treated with recombinant granulocyte colony-stimulating factor for 3 days. Absolute neutrophil counts increased nearly four-fold within 48 hours; maximal values were recorded on the ninth day after the infusion was started. Total leukocyte counts subsequently decreased but remained in the normal range. It appears that recombinant human granulocyte colony-stimulating factor promotes a rapid increase in circulating neutrophils in these patients despite the possible presence of a circulating preeclampsia-associated inhibitor of neutrophil production.

Dopaminergic regulation of a transfected preproenkephalin promoter in primary rat astrocytes in vitro and in vivo.

The clinical benefit of transplantation therapies utilizing genetically modified cells could be enhanced if expression of engineered genes was regulated by clinically useful pharmacological agents. Toward this end, we examined pharmacologic effects on the expression of hybrid gene constructs transfected into primary rat striatal astrocytes. These astrocytes are known to express receptors for the neurotransmitter dopamine (DA). In vitro, we found that expression of a transiently transfected human ppEnk promoter-driven chloramphenicol acetyltransferase (CAT) reporter construct was induced by DAergic agonists, as much as 20-fold. This induction was blocked by a DA receptor antagonist. The same concentration of DA also increased the endogenous rat ppEnk mRNA, by > 2-fold. In vivo, regulation of CAT expression by DA was tested by implanting the genetically modified astrocytes into the normal striatum and the contralateral striatum which had > 95% DA depletion induced by a previous 6-hydroxy-DA lesion of the substantia nigra. As hypothesized on the basis of the in vitro data, CAT activity on the lesioned side, where the stimulating effect of endogenous DA was lacking, was 30% lower than on the control side where the normal DA content was present. The data suggest that control of the enkephalin gene in astrocytes may involve second messenger pathways activated by DA receptors. Moreover, the evidence that clinically applicable drugs can regulate inducible genes introduced into the brain by astrocyte implantation is of potential importance in development of therapeutic strategies.

Feeding practices for infants weighing less than 1500 G at birth and the pathogenesis of necrotizing enterocolitis.

This article examines clinical issues regarding gut maturation, gut colonizatiion, gut luminal starvation, a germ-free gut, and the role of enteral intake in the pathogenesis of necrotizing enterocolitis (NEC) in very low birth weight neonates and micropremies. NEC is identified as the final common pathway for a variety of etiologic mechanisms, only one of which is consistent with the enteral-based theory of NEC. The technique of minimal enteral intake ("gut priming") is discussed as a strategy to maintain the normal ontological processes of the developing gut ex utero. A combination of enteral plus parenteral intake is described to achieve nutritional goals.

Tissue-specific DNaseI hypersensitivity regions are located in the 5'-region of the rat preproenkephalin gene.

Extracellular stimuli affecting mechanisms of transcriptional control of the preproenkephalin gene link stimulus-secretion-synthesis coupling to transmitter phenotypic expression. In order to define distal and proximal genomic regions that might participate in transcriptionally active protein-DNA interactions, DNaseI hypersensitivity assays were conducted. Four DNaseI-hypersensitive regions were identified at -3000, -2500, -1800, and -200 base pairs (bp) relative to the somatic start site. The appearance of a site over the proximal promoter/enhancer region (-200 bp) is of interest since we and others have published a detailed footprint and functional analysis of this region using rat tissues. While liver (which does not express proenkephalin mRNA) displays essentially no hypersensitivity here, adrenal gland preparations show a strong signal and striatal tissue a significantly weaker signal. Cholinergic induction of proenkephalin mRNA was not associated with a change in hypersensitivity pattern. These data suggest that chromatin structure may have an influence on preproenkephalin transcriptional control and that cholinergic-inducible cis-acting DNA elements may reside within subdomains of at least these four hypersensitivity sites. Understanding the basis of this open chromatin structure should aid in identifying genomic mechanisms involved in tissue-specific expression and suggest avenues of future study.

Primary sequence of -1436 to +53 bp of the rat preproenkephalin gene putative Z-DNA and regulatory motifs.

We report novel sequence data extending -1436 bases 5' of the rat proenkephalin gene start site known as E4. We noted an interesting stretch of 58 bases of alternating pyrimidines that lies immediately adjacent to 71 bases of an alternating purine-pyrimidine Z-DNA-like sequence that lies between -694 bp and -566 bp. Multiple sequence homologies to putative cis-acting regulatory factor binding sites were identified by a computer aided sequence search.

Regulation of adrenomedullary preproenkephalin mRNA: effects of hypoglycemia during development.

To further evaluate whether transsynaptic mechanisms account for stress-induced changes in adrenomedullary preproenkephalin mRNA (ppEnk mRNA), neonatal rats were made hypoglycemic at a time when synapses are non-functional (less than 10 days postnatal age). While ppEnk mRNA in medullae from adult rats increased as much as 60-fold in this paradigm (insulin 10 U/kg), ppEnk mRNA levels in the newborn increased only 1.6-fold (insulin 20 U/kg). To evaluate whether postsynaptic cholinergic pathways of the neonatal adrenal medulla were functional, we treated 5-day-old pups with cholinergic agonists (nicotine [1 mg/kg, s.c., q 12 h] + carbachol [1.7 mumol/kg, s.c., q 12 h x 4 days]). Combined cholinergic agonist treatment augmented enkephalin prohormone and peptide levels up to 3-fold (P less than 0.05). To determine whether the blunted response to hypoglycemia in the newborn resulted from a deficiency in functional transsynaptic activity, synapses were matured using thyroid hormone pretreatment (postnatal days 2 and 3) before hypoglycemic stress. Hypoglycemia now caused a 40-fold increase in adrenomedullary ppEnk mRNA levels only in the T3/insulin treated group. To exclude other secondary effects of hypoglycemia (eg. hormonal, or insulin treatment-dependent), intracellular glycopenia was produced in the presence of secondary hyperglycemia by injecting adult rats or pups with 2-deoxyglucose (500 mg/kg). Similar to the insulin-hypoglycemia group, a large increase in adrenomedullary ppEnk mRNA resulted in the adult but not in the 5-day-old neonatal adrenal medullae. We conclude that enkephalin biosynthesis, like co-stored catecholamines, is induced by a transsynaptic process.(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible preproenkephalin DNA binding proteins in the rat striatum.

The molecular manifestation of whole animal and cellular adaptive responses results from stimulus-secretion-synthesis coupling to selected genes. Effects are mediated through receptor-linked, signal-transduction pathways involving trans-acting nuclear proteins binding to cis-acting DNA regulatory elements. In the current report we have identified those striatal nuclear factors that footprint to the 5' region of the rat preproenkephalin (ppEnk) gene and how they change following ppEnk induction. These studies demonstrate binding of Spl-like factors, as well as members of the Jun and Fos families of proteins. Unique footprints were also noted in the ppEnk-induced state in the region of GGTGGGGGAGCCTCCGG (-91 to -175) overlapping the consensus binding site for the brain-specific transcription factor beta; a functional DNA element for the rat ppEnk gene. These observations illustrate that DNA-protein interactions both before and after gene induction can be resolved even in complex primary structures of the central nervous system.

Cholinergic agonist-induced binding of adrenomedullary nuclear proteins to the rat preproenkephalin promoter.

Cholinergic receptors are well-recognized modulators of transmitter release and biosynthesis with newly identified actions mediated through trans-acting DNA binding proteins affecting gene expression at cis-acting regulatory elements. In the case of the rat preproenkephalin (ppEnk) gene, we have identified changes in ppEnk DNA binding proteins detected by gel mobility shift assay that correlate with a cholinergic induction of ppEnk RNA levels. Antibody competition experiments implicate Fos-like and Jun-like transcription factors in this phenomenon. Methylation interference footprinting identified two cholinergic-inducible enhancements in the regions -155 to -149 and -185 to -173 (BETA1 site). An additional footprinted region at the BETA2 site (-137 to -132) was unaffected by cholinergic agonist treatment. These footprint features are significantly different from those obtained from the striatum after identical cholinergic drug treatments. We speculate that basal as well as cholinergic agonist responsive transcription factors account for tissue-specific differences in ppEnk gene expression. Further characterization of these cholinergic agonist-inducible events will require functional analysis of footprinted domains to evaluate the in vivo role of these putative gene regulatory elements.

Bimodal regulation of adrenal opiate peptides by cholinergic mechanisms.

Physiologic stressors increase trans-synaptic impulse activity and result in adrenal catecholamine release and biosynthesis. To determine the effects of stress on the co-localized opiate peptide system, rats were cold stressed at 4 degrees C. While cold stress slightly decreased enkephalin levels, a more severe stress (wetting and cold) increased enkephalin levels by 95%. Examining trans-synaptic-cholinergic mechanisms, treatment with either nicotinic or muscarinic agonists alone resulted in no change in adrenal enkephalin content. However, treatment with both nicotinic and muscarinic agonists together resulted in a three-fold rise in enkephalin levels. To further examine cellular mechanisms, medullae were explanted in the presence of agents that increase second messenger cyclic nucleotide levels. Treatments that increase the levels of cAMP, the cyclic nucleotide associated with nicotinic receptor activation, prevented the rise in medullary enkephalin relative to control explants. In contrast, treatments that increased cGMP levels, the cyclic nucleotide associated with muscarinic receptor activation, had no effect on enkephalin content compared to control explants. However, in the presence of both forskolin (10 microM) plus db-cGMP (5 mM), enkephalin content rose three-fold over control explants. These data suggest that, distinct from catecholamine pathways, enkephalin levels can be positively or negatively regulated by the severity of a stressful stimulus, by cholinergic receptor mechanisms and by an interaction of cyclic nucleotide second-messenger pathways.

Preproenkephalin DNA-binding proteins in the rat: 5' flanking region.

Various extracellular signals (i.e. transmitters, hormones, growth factors, etc.), together with their respective second-messenger pathways, regulate transmitter biosynthesis and neuronal function by altering gene expression. In this study we validated a protocol for isolating rat striatum and adrenal medullary nuclei for the purpose of extracting, identifying, and characterizing, nuclear regulatory factors which may serve a functional role in signal-transduction processes. Through gel retardation studies using a 299 base pair (bp) XmnI-SacI 32P-labeled probe (derived from the 5' untranslated region of the rat preproenkephalin gene), we show that different patterns of retained bands result from nuclear extracts derived from rat adrenal medulla and striatum (as well as from other tissue). These tissue differences may have biological significance since rat adrenal medullae have low basal enkephalin levels while the striatum has high levels of this peptide and its respective mRNA. Additionally, certain retained bands were common to both cytosolic and nuclear compartments, suggesting binding factors may be located in either cell space. An initial biochemical characterization of these factors was also undertaken. Generally, salt levels of 100 mM or more reduced factor binding while 10-50 mM sodium ion levels showed preferentially enhanced bands. Binding activity appeared optimal at pH 6.8. As all retained bands were abrogated by proteinase K treatment, these factors appear to have a significant protein component. Finally, of particular interest is that this 299 bp region contains many sequences showing over 80% sequence identity with several previously characterized transcriptional control elements (i.e. cAMP and phorbol ester inducible enhancers, GCN4, AP1, Sp1, CCAAT binding factor, ATF, and AP2). If binding is confirmed (footprint analysis) and function validated (transfection studies), the evolutionary significance of the apparent presence of gene regulatory sequences and functional element divergence of the DNA region between different species can be evaluated.