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Biomedical subjects

D G Johnson

Publications and source records attributed to D G Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1993

cDNA cloning and expression of guinea pig neutrophil attractant protein-1 (NAP-1). NAP-1 is highly conserved in guinea pig.

cDNA for neutrophil attractant protein-1 (NAP-1, also known as IL-8) was cloned from Con A-stimulated guinea pig spleen cells with human NAP-1 cDNA as a probe. Guinea pig NAP-1 cDNA is composed of 1433 bp with an open reading frame which encodes for a 101-amino-acid protein. Guinea pig NAP-1 had 70% amino acid sequence similarity to human NAP-1, which was much higher than a similarity between human and guinea pig monocyte chemoattractant protein-1 (MCP-1) (56%). Nucleotide sequence similarity within the coding region was 75%. To confirm its biological activity in guinea pig, recombinant guinea pig NAP-1 was expressed in COS-7 cells then purified. N-terminal sequence analysis gave two different N-termini at position 23 (Met) or 24 (Val). The two proteins showed their peak activity for guinea pig neutrophils at the concentration of 1 microgram/ml (10-7 M). Despite its high similarity to human NAP-1, the responsiveness of human neutrophils to guinea pig NAP-1 was minimum. Recombinant guinea pig NAP-1 caused strong neutrophil infiltration after intradermal injection into guinea pig skin. Since guinea pig is classified as a rodent, it was of interest to know whether human NAP-1 cDNA hybridizes to genomic DNA of other rodents such as mouse or rat, in which a NAP-1 homologue has not been found. Under low stringency conditions, human NAP-1 cDNA hybridized to human, rabbit, and guinea pig DNA, but not to mouse or rat DNA. Unlike NAP-1, human MCP-1 cDNA hybridized to genomic DNA of rabbit, guinea pig, mouse, and rat; MCP-1 cDNA have been cloned from these species. The apparent absence of a NAP-1 gene in mouse or rat makes this chemoattractant unique among the members of the protein family to which NAP-1 and MCP-1 belong.

Amino Acid Sequence↗
PubMed1993

Expression of transcription factor E2F1 induces quiescent cells to enter S phase.

Several lines of evidence implicate the E2F transcription factor as an important component of cell proliferation control. First, E2F binding sites are found in the promoters of genes responsive to proliferation signals and the level of E2F binding activity increases at a time when many of these genes are activated. Second, the tumour suppressor protein Rb, as well as the related p107 protein, complexes with E2F, resulting in an inhibition of E2F transcriptional activity. Third, oncogenic products of the DNA tumour viruses can dissociate these E2F complexes. We provide here direct evidence that E2F is involved in cellular proliferation control. Specifically, we demonstrate that overexpression of the E2F1 complementary DNA can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus E1A gene. Moreover, microinjection of the E2F1 cDNA into quiescent cells can induce S-phase entry, whereas two E2F1 mutants, which are unable to transactivate the DHFR and TK promoters, are unable to induce S phase. We conclude that the E2F transcription factor plays an important role in progression into S phase and that this probably coincides with its capacity to stimulate transcription.

Adenoviridae↗
PubMed1993

Morphologic basis for loss of regulated insulin secretion by isolated rat pancreatic islets.

Laboratories engaged in secretory studies of rat pancreatic islets often encounter high baseline insulin secretion with poor secretory response to secretagogues, such as glucose. The specific morphologic abnormalities that accompany this unregulated release have not been described. We isolated islets comparing two approaches. Both used stationary digestion with collagenase. In method I, we distended the biliary duct extracorporeally with collagenase and minced the pancreas after a 28 min digestion (37 degrees C). In method II, we distended the pancreas intracorporeally and digested for 40 min without mincing. Both methods utilized a similar collagenase concentration (2 micrograms/ml in Hank's balanced salt solution (HBSS). Both methods yielded over 300 islets/rat. Islets from both methods appeared intact, when viewed under the dissecting microscope. We found that adequate secretion from incubated islets was evoked with method I, i.e., low basal insulin levels at low glucose (3.3 mM), tripling at 11.0 mM glucose, and nearly quadrupling in response to higher glucose (16.7 mM). In contrast, method II was characterized by high basal levels without response to higher glucose. Ultramicroscopic examination of islet B cells in method I revealed normal cytological features, while B cells in method II showed marked degranulation, profiles of swollen endoplasmic reticulum, and swollen mitochondria. Morphometric analysis of B cells confirmed quantitatively a decrease in secretory granule density and mitochondrial enlargement in method II compared to method I. Anatomic changes, largely confined to the B cells of islets may account for functional alterations of responses. Defects cannot be predicted from gross appearance of islets.

Animals↗
PubMed1993

New glucagon analogues with conformational restrictions and altered amphiphilicity: effects on binding, adenylate cyclase and glycogenolytic activities.

In an effort to obtain highly potent glucagon antagonists, we have investigated glucagon (1) structure-function relationships utilizing the following design principles: (1) structural changes known to lead to partial agonist activities; (2) conformational restrictions; (3) changes in the conformational probabilities of the primary sequence; and (4) increased amphiphilicity. In this report we present the total synthesis, purification, receptor binding, adenylate cyclase activity, in vivo glycogenolytic activity and CD spectrum of the following four glucagon analogues: [Ahx17,18]glucagon (2), [D-Phe4,Tyr5, 3,5-diiodo-Tyr10,Arg12,Lys17,18,Glu21]glucagon (3), [Asp9,Lys12,Lys17,18,Glu21]glucagon 4, and [Glu15,Lys17,18]glucagon 5. Compound 2 binds exclusively to the high affinity receptor and compound 3 was a highly potent antagonist with respect to adenylate cyclase activity. Analog 4 showed distinct biphasic binding (IC50 5.6 nM and 630 nM), with only the low affinity binding leading to adenylate cyclase activity. Furthermore in analogue 5 receptor binding and adenylate cyclase activity were dissociated by a factor of 5. The results are consistent with a multistep binding mechanism in which glucagon interacts first nonspecifically with the anisotropic interphase of the cell membrane, followed by a conformational transition which occurs in the sequences 10-14 and 15-18 when the membrane bound peptide binds to its receptor.

Adenylyl Cyclases↗
PubMed1993

A genetic analysis of the E2F1 gene distinguishes regulation by Rb, p107, and adenovirus E4.

The cellular transcription factor E2F appears to be a target for the regulatory action of the retinoblastoma tumor suppressor gene product. The recent isolation of the E2F1 cDNA clone, which encodes a polypeptide with properties characteristic of E2F, has now allowed a more detailed analysis of the regulation of E2F function by Rb as well as the Rb-related p107 protein and the adenovirus 19-kDa E4 gene product. Previous experiments have shown that each of these regulatory proteins can modulate the activity of cellular E2F. We find that each of these regulatory events can be mediated through the E2F1 product. Moreover, an examination of various E2F1 mutations reveals distinct specificities for these regulatory proteins. For instance, the ability of E4 to alter E2F1 function is dependent upon sequences within a putative leucine repeat of E2F1 as well as within the C-terminal acidic domain. In contrast, the leucine repeat element was not important for Rb- or p107-mediated inhibition of E2F1 activity. Although the C-terminal acidic domain of E2F1, previously shown to be important for Rb binding, appears to be a site for regulation of E2F1 by Rb and p107, point mutations within this region distinguish recognition by Rb and p107. These results underscore the complexity of E2F regulatory interactions and also demonstrate a qualitative distinction in the interactions of Rb and p107 with E2F1, perhaps reflective of functional differences.

Adenovirus E4 Proteins↗
PubMed1993

Tissue-specific noradrenergic activity during acute heat stress in rats.

The purpose of this study was to determine whether nonexertional heat stress alters the behavior of internal organ and skeletal muscle sympathetic nervous system (SNS) activity in unrestrained conscious rats. Norepinephrine (NE) synthesis was blocked with alpha-methyl-p-tyrosine, and the rate of decline in tissue NE concentration after synthesis blockade was used to estimate SNS activity in the left ventricle, kidney, liver, adrenal gland, and soleus and extensor digitorum longus muscles of the hindlimb. Male Sprague-Dawley rats (250-320 g) were maintained in thermoneutral control conditions (ambient temperature = 24 degrees C, n = 10) or exposed to an ambient temperature of 42 degrees C until a colonic temperature (Tc) of 39.5 or 41.0 degrees C (n = 10 each) was attained. During heating, as Tc rose from control levels (approximately 38 degrees C) to 41.0 degrees C, mean arterial pressure and heart rate increased from 120 +/- 3 to 141 +/- 3 mmHg and from 381 +/- 4 to 420 +/- 5 beats/min, respectively (P < 0.05). There was a strong trend for increased NE turnover rates in the left ventricle, liver, and adrenal gland in NE synthesis-blocked rats attaining a Tc of 39.5 degrees C compared with the normothermic values, whereas the turnover rate in the kidney was significantly elevated at this level of hyperthermia (126%) vs. the control condition. In animals heated to a Tc of 41.0 degrees C, the NE turnover rate was markedly increased in the left ventricle (590%), kidney (531%), liver (262%), and adrenal gland (602%) compared with normothermic control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Age and gender influence muscle sympathetic nerve activity at rest in healthy humans.

Muscle sympathetic nerve activity at rest increases with age in humans. The respective influences of the aging process per se and gender on this increase and whether age and gender effects on muscle sympathetic nerve activity can be identified with plasma norepinephrine concentrations, however, have not been established. To examine these issues, nine young women (aged 24 +/- 1 years; mean +/- SEM), eight young men (aged 26 +/- 1 years), seven older women (aged 63 +/- 1 years), and eight older men (aged 66 +/- 1 years) were studied. All were healthy, normotensive (blood pressure < 140/90 mm Hg), nonobese (< 20% above ideal weight), unmedicated, nonsmokers engaged in minimal to recreational levels of chronic physical activity. Arterial blood pressure (manual sphygmomanometry, brachial artery), heart rate, muscle sympathetic nerve activity (peroneal microneurography), and antecubital venous plasma norepinephrine concentrations (radioenzymatic assay) were determined during quiet supine resting conditions. Body weight was higher in men, but there were no age-related differences, whereas estimated body fat (sum of skinfolds) was higher in women and in the older groups (p < 0.05). Estimated daily energy expenditure, arterial blood pressure, and heart rate were not different among the groups. Both muscle sympathetic nerve activity burst frequency and burst incidence at rest were progressively higher in the young women, young men, older women, and older men (10 +/- 1 versus 18 +/- 2 versus 25 +/- 3 versus 39 +/- 5 bursts/min and 16 +/- 1 versus 30 +/- 4 versus 40 +/- 3 versus 61 +/- 6 bursts/100 heartbeats, respectively; all p < 0.05 versus each other).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Lupins (Lupinus albus) as a protein supplement for lactating Holstein dairy cows.

Three experiments were conducted to determine optimal substitution of lupin for soybean meal in dairy cattle diets; the effects of whole versus ground lupins and the effects of whole lupins versus whole soybeans on milk production were examined. In the first experiment, 57 cows were assigned randomly to treatments in blocks of five by calving order within parity. Treatments began 22 d postpartum and continued through d 140. Diets were isonitrogenously balanced using soybean meal as control: lupins replaced 0, 25, 50, 75, and 100% of supplemental protein. Cows that consumed 75% of supplemental protein as lupins produced more 3.5% FCM and fat than cows that consumed the control diet. In a second experiment, 30 primiparous cows were assigned randomly by calving order to either ground or whole lupins (60% of supplemental protein). Treatments began 32 +/- 3 d after calving and lasted for 84 d. Cows that consumed ground lupins produced more milk and 3.5% FCM than cows that consumed whole lupins. In the third experiment of 84 d, 28 primiparous midlactation cows were assigned randomly to diets containing whole lupins or whole soybeans to supply 1 kg of supplemental protein. Cows fed whole lupins produced milk with higher fat content, more 3.5% FCM, and more fat than cows consuming whole soybeans. Lupins are an acceptable supplemental protein source for dairy cows.

Animal Nutritional Physiological Phenomena↗
PubMed1992

Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processings, and complete amino acid sequences.

The MN strain of human immunodeficiency virus type 1 was grown in H9 cells, concentrated by centrifugation, and disrupted, and proteins were purified by reversed-phase high-pressure liquid chromatography. Complete amino acid sequences were determined for the mature Gag proteins, showing natural proteolytic cleavage sites and the order of proteins (p17-p24-p2-p7-p1-p6) in the Gag precursors. At least two sequence variants of p24 and eight sequence variants of p17 were detected. The two most abundant variants of p24 and p17 represented at least 50% +/- 5% and 20% +/- 5% of their totals, respectively. These data suggest heterogeneity in the virus population, with 50% of the total virus containing the most abundant forms of p17 and p24 and 20% of the virus containing the second most abundant forms. The Gag precursors of these suggested viruses differ from each other by only 3 amino acid residues but differ from the precursors predicted by the published MN proviral DNA sequence by 10 residues. Electrospray ionization mass spectrometry analysis of the purified p24 forms showed that the measured molecular weight of the protein was 200 +/- 50 atomic mass units greater than the calculated molecular weight. The source of additional mass for the p24 forms was not determined, but the observation is consistent with previous suggestions that the protein is phosphorylated. Greater than 98% of the total recovered p17 was myristylated at the N-terminal glycine residue, and the measured molecular weights (as determined by electrospray ionization mass spectrometry) of the most abundant forms were within 3 atomic mass units of the calculated molecular weights (15,266).

Amino Acid Sequence↗
PubMed1992

Sympathoadrenal-circulatory regulation of arterial pressure during orthostatic stress in young and older men.

Our purpose was to test the hypothesis that human aging alters sympathoadrenal-circulatory control of arterial blood pressure during orthostasis. Plasma catecholamine and hemodynamic adjustments to two different forms of orthostatic stress, lower body suction (-10 to -50 mmHg) and standing, were determined in 14 young (26 +/- 1 yr) and 13 older (64 +/- 1) healthy, normally active men. During quiet supine rest, cardiac output tended to be lower and systemic vascular resistance higher in the older men, but no other differences were observed. On average, arterial blood pressure was well maintained during both forms of orthostasis in the two groups; the older men actually demonstrated better maintenance of pressure (P < 0.05) and a lesser incidence of orthostatic hypotension than the young men during lower body suction. Despite a blunted reflex tachycardia during orthostatic stress (P < 0.05), cardiac output tended to decrease less in the older men because of a smaller decline in stroke volume (P < 0.05, suction only), whereas the reflex increases in systemic vascular resistance were not different in the two groups. The whole forearm vasoconstrictor response tended to be attenuated in the older men during lower body suction, but was identical in the two groups with standing. Forearm skin vascular resistance was unaltered during lower body suction in both groups. Orthostasis-evoked increases in antecubital venous plasma norepinephrine concentrations were similar in the young and older men, whereas little or no increases in plasma epinephrine concentrations were observed in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1992

Augmented forearm vasoconstriction during dynamic exercise in healthy older men.

BACKGROUND: We tested the hypothesis that the nonactive limb vasoconstriction evoked during large-muscle dynamic exercise becomes augmented with aging in humans. METHODS AND RESULTS: Sixteen young control subjects (age, 26 +/- 1 year) and twelve older (65 +/- 1 year) healthy men with similar chronic physical activity levels were studied during supine leg cycling exercise. Both peak work load (1,100 +/- 60 versus 1,400 +/- 40 kpm/min) and peak O2 uptake (1.85 +/- 0.10 versus 2.38 +/- 0.07 l/min) were lower in the older men (p < 0.05). There were no differences in the two groups under conditions of quiet supine (basal) rest. During cycling for 5 minutes each at mild, moderate, and heavy submaximal intensities (approximately 45%, 65%, and 85% of peak O2 uptake), the increases in arterial blood pressure generally were similar in the young and older subjects; however, heart rate rose less in the older men (p < 0.05). Whole forearm blood flow (venous occlusion plethysmography) was lower and vascular resistance was higher (approximately 55-90%) in the older men at all loads (p < 0.05), but the steady-state forearm skin blood flow responses (laser Doppler velocimetry) were not different in the two groups. The increases in antecubital venous norepinephrine concentrations were greater in the older men at each work load (p < 0.05), although the plasma epinephrine responses were similar in the two groups. In other studies, 1) peak whole forearm reactive hyperemia and vascular conductance after sustained circulatory arrest (ischemia) were slightly (approximately 20%) but not significantly lower in the older men and 2) the forearm vasoconstrictor and plasma norepinephrine responses to a nonexercise sympathoexcitatory stimulus (limb immersion in ice water) tended to be blunted in the older men. CONCLUSIONS: During brief, submaximal, large-muscle dynamic exercise, healthy older men demonstrate augmented forearm vasoconstriction that is probably caused by greater constriction of skeletal muscle resistance vessels; this appears to be mediated, at least in part, by increased sympathetic outflow. These altered sympathetic vasoconstrictor adjustments do not represent a nonspecific hyperresponsiveness to acute stress with human aging. Finally, the regulation of arterial blood pressure appears to be normal in these healthy older men.

Adult↗
PubMed1991

The mean duration of gastroesophageal reflux during sleep as an indicator of respiratory symptoms from gastroesophageal reflux in children.

The coexistence of gastroesophageal reflux (GER) and respiratory symptoms has been reported in children, but identifying a reliable indicator of reflux-induced respiratory symptoms has been difficult. The mean duration of reflux episodes during sleep (ZMD), extracted from a 18- to 24-hour esophageal pH recording, has been presented previously as a reliable correlate of respiratory symptoms caused by GER in infants. To reexamine the accuracy of the ZMD we report 519 consecutive children with respiratory symptoms (aged 1 week to 15 years) who were referred for documentation of GER by 18- to 24-hour esophageal pH monitoring. Follow-up information was obtained on 388 patients 1 to 115 months later (mean, 23 months). From the follow-up information, 259 children could be reliably classified; 128 as having reflux-related and 131 as having reflux-unrelated respiratory symptoms. None of the 131 children with reflux-unrelated respiratory symptoms had a prolonged ZMD (greater than 3.8 minutes). In comparison, 119 of the 128 children (93%) with reflux-related respiratory symptoms had a prolonged ZMD (P less than .001). The nine children who had a normal ZMD and improvement in their respiratory symptoms with the successful control of GER had advanced esophagitis (n = 4), central apnea (n = 3), or associated disorders in which respiratory symptoms improved with the avoidance of oral feedings (n = 2). In conclusion, the ZMD was confirmed to correlate directly with both the presence and absence of respiratory symptoms caused by GER in children.

Adolescent↗
PubMed1991

The risk of sudden infant death from gastroesophageal reflux.

Gastroesophageal reflux (GER) has been a suspected cause of infant deaths and sudden infant death syndrome (SIDS). We examined our 10-year experience with 499 consecutive infants 6 months of age or less who had extended (18 to 24 hours) esophageal pH monitoring performed to evaluate for GER. The data extracted from the esophageal pH records included the pH score, the pattern of GER (type I, II, or III), and the mean duration of reflux during sleep (ZMD). All infants were followed to determine the occurrence and cause of death during the first year of life. Of the 19 deaths found in the series, three were classified as SIDS and two were in-hospital deaths caused by reflux-induced aspiration. All five of these infants who died had a prolonged ZMD (greater than 3.8 minutes) and received either basic medical (n = 4) or no (n = 1) antireflux therapy. Four infants also had the type I pattern of GER. There was a 9.1% (4/44; 95% confidence limits, 2.5% to 21.7%) incidence of reflux-related or SIDS deaths in infants with type I GER and a prolonged ZMD who were treated nonoperatively, compared with none (0/83, P = .03) in the same group of infants treated with antireflux surgery. The incidence of SIDS was higher in infants with type I GER and a prolonged ZMD who were treated nonoperatively (3/44, 6.8%) compared with all other infants treated nonoperatively (0/265, 0%; P = .003).(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1991

Gastroesophageal reflux: a significant association with central nervous system disease in children.

An association between gastroesophageal reflux (GER) and central nervous system (CNS) disease has been suggested, but not defined clearly in children. We report on 613 children (ages 1 week to 16 years; mean, 16 months) studied at three institutions who were referred to the pediatric surgical service for documentation of GER by an abnormal pH score derived from 18- to 24-hour esophageal pH monitoring. Follow-up (range, 1 to 115 months; mean, 25 months) was obtained in 368 patients. One hundred thirty-two children had CNS disease documented prior to the evaluation for GER. In children older than 1 year, there was an increased incidence of GER (31/45, 69%) in those children with CNS disease compared with those without CNS disease (38/81, 47%; P = .014). On follow-up, only 4.6% (14/282) of children who were not diagnosed initially with CNS disease were found later to have overt CNS disease. There was no significant difference in the prevalence of newly diagnosed CNS disease in children with and without GER in long-term follow-up. In conclusion, in the population of children referred to the pediatric surgeon for evaluation of GER, children older than 1 year with CNS disease are at high risk to have associated GER documented by extended esophageal pH monitoring. Despite this association, those children with GER and no obvious CNS disease do not appear to be at increased risk to develop CNS disease.

Adolescent↗
PubMed1991

Hyperoxia lowers sympathetic activity at rest but not during exercise in humans.

The primary aim of this study was to determine the influence of systemic hyperoxia on sympathetic nervous system behavior at rest and during submaximal exercise in humans. In seven healthy subjects (aged 19-31 yr) we measured postganglionic sympathetic nerve activity to skeletal muscle (MSNA) in the leg, antecubital venous norepinephrine concentrations, heart rate, and arterial blood pressure during normoxic rest (control) followed by 3- to 4-min periods of either hyperoxic (100% O2 breathing) rest, normoxic exercise (rhythmic handgrips at 50% of maximum force), or hyperoxic exercise. During exercise, isocapnia was maintained by adding CO2 to the inspirate as necessary. At rest, hyperoxia lowered MSNA burst frequency (12-42%) and total activity (6-42%) in all subjects; the average reductions were 25 and 23%, respectively (P less than 0.05 vs. control). Heart rate also decreased during hyperoxia (6 +/- 1 beats/min, P less than 0.05), but arterial blood pressure was not affected. During hyperoxic compared with normoxic exercise, there were no differences in the magnitudes of the increases in MSNA burst frequency or total activity, plasma norepinephrine concentrations, or mean arterial blood pressure. In contrast, the increase in heart rate during hyperoxic exercise (13 +/- 2 beats/min) was less than the increase during normoxic exercise (20 +/- 2 beats/min; P less than 0.05). We conclude that, in healthy humans, systemic hyperoxia 1) lowers efferent sympathetic nerve activity to skeletal muscle under resting conditions without altering venous norepinephrine concentrations and 2) has no obvious modulatory effect on the nonactive muscle sympathetic nerve adjustments to rhythmic exercise.

Adult↗