bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro and in vivo responses to short‐term recombinant human insulin‐like growth factor‐1 (IGF‐I) in a severely growth‐retarded girl with ring chromosome 15 and deletion of a single allele for the type 1 IGF receptor gene
Authors: De Lacerda Luiz , Carvalho Julienne A. R. , Stannard Bethel , Werner Haim , Boguszewski Margaret C. S. , Sandrini Romolo , Malozowski Saul N. , LeRoith Derek , Underwood Louis E.
Published: May 2003

OBJECTIVES

Patients with single allele defects in the gene encoding the type 1 IGF receptor have been reported to have growth failure, but fibroblasts from affected patients have not exhibited insensitivity to the effects of IGF‐I in vitro. The in vitro and in vivo responses to short‐term recombinant human IGF‐I (rhIGF‐I) in a severely growth‐retarded girl with ring chromosome 15 and deletion of a single allele for the type 1 IGF receptor gene have been investigated.

DESIGN AND PATIENT

The child exhibited prenatal and severe post‐natal growth failure, and delayed psychomotor development. Southern blotting revealed a 50% reduction in IGF‐I receptor DNA, and in an RNase protection assay (RPA), a quantitatively similar reduction in steady‐state mRNA for type 1 IGF receptor. rhIGF‐I was administered in graded doses of 40, 60 and 80 μg/kg twice daily by subcutaneous injection for periods of 2–2.5 days each.

RESULTS

During rhIGF‐I treatment, mean urinary nitrogen excretion was unchanged and urinary calcium rose to 60% greater than in the pre‐treatment period. rhIGF‐I injections produced only a modest decrease in indices of GH secretion, assessed by frequent (every 20 min) sampling over periods of 12 h. There was no significant difference between the mean GH concentrations during rhIGF‐I treatment (5.32 ± 6.2 mU/l) compared with that before rhIGF‐I treatment (8.46 ± 10.2 mU/l). Mean IGFBP‐3‐values were increased (4.5 mg/l before vs. 5.4 mg/l during rhIGF‐I). TSH values after injection of TRH were not significantly reduced by IGF‐I (mean of all values, 18.6 mU/l vs. 15.5 mU/l during rhIGF‐I treatment). In vitro binding of radiolabelled IGF‐I to the patient's fibroblasts was less than that bound by control fibroblasts (patient, 0.69% binding by 248 000 cells, vs. 1.41% binding by 260 000 fibroblasts from an age‐matched control). However, the patient's fibroblasts exhibited a growth response in vitro to the addition of IGF‐I in a fashion similar to that of control fibroblasts.

CONCLUSIONS

These studies show evidence in each of the indices examined of in vivo resistance to IGF‐I and suggest that the growth retardation observed in such patients may be the direct result of the absence of one of the alleles encoding the type 1 IGF receptor.
Serum leptin and body composition in children with familial GH deficiency (GHD) due to a mutation in the growth hormone‐releasing hormone (GHRH) receptor
Authors: De A. Barretto Elenilde S. , Gill Matthew S. , De Freitas Maria E. S. , Magalhães Marta M. G. , Souza Anita H. O. , Aguiar‐Oliveira Manuel H. , Clayton Peter E.
Published: May 2003

OBJECTIVE

The relationship between GH, body composition and leptin in children remains ill‐defined. We have therefore examined the impact of severe GH deficiency (GHD) due to a mutation in the GHRH receptor on serum leptin concentrations and body composition in childhood.

PATIENTS

12 affected children and young people (GHD) (4 M:8F, age 5.4–20.1 years, 8 Tanner stage (TS) 1–2, 4 TS 3–5) and 40 healthy controls (C) from the same region (13 M:27F, age 5.3–18.4 years, 20 TS 1–2, 20 TS 3–5).

METHODS

Percent body fat was determined by infra‐red interactance, from which the amounts of fat mass (FM, kg) and fat free mass (FFM, kg) were derived. Serum leptin concentrations were measured in a single fasted, morning serum sample and results expressed as a concentration and as leptin per unit fat mass (L/FM, ng/ml/kg). To control for differences in sex and pubertal maturation, leptin standard deviation scores (leptin SDS) were calculated using normative data from UK children.

RESULTS

FFM was significantly lower in GHD children than in controls (TS 1–2 P < 0.05, TS 3–5 P < 0.001). FM did not differ significantly between the two groups. Serum leptin concentrations, leptin per unit fat mass and leptin SDS were significantly elevated in GHD children both peripubertal and pubertal compared with controls. Using all subjects, stepwise multiple linear regression with FM, FFM, age, puberty and sex as explanatory variables and leptin concentration as the dependent variable indicated that 59% of the variability in leptin could be accounted for by FM (+ , 45%), FFM (− , 9%) and sex (+ , 5%) (P < 0.001). However on inclusion of GH deficiency (coded GHD = 1, control = 2) as an explanatory variable 73% of the variability in leptin was explained by FM (+ , 45%), GHD (− , 22%) and sex (+ , 6%) (P < 0.001).

CONCLUSIONS

These data indicate that severe GH deficiency in children is associated with elevated leptin concentrations, irrespective of sex or pubertal stage. This increase is not associated with differences in fat mass but is related to reduced fat free mass in GH deficiency. Furthermore in this population there may be an additional effect of GH deficiency on leptin, independent of the influences of sex and body composition.
High serum leptin levels in children with type 1 diabetes mellitus: contribution of age, BMI, pubertal development and metabolic status
Authors: Luna Reyes , Garcia‐Mayor Ricardo V. , Lage Mary , Andrade M. Amalia , Barreiro Jesús , Pombo Manuel , Dieguez Carlos , Casanueva Felipe F.
Published: May 2003

OBJECTIVE

Children with diabetes mellitus are prone to develop obesity and to experience a delay in onset of the pubertal process. In order to understand the role of leptin in these abnormalities, serum leptin levels were analysed in children with type 1 diabetes mellitus.

SUBJECTS

Twenty diabetic girls, 23 diabetic boys and 66 healthy children (selected from a reference population of 706 normal children), age‐, sex‐ and BMI‐matched with diabetic patients, were studied.

MEASURMENTS

Standing height, weight and BMI were determined in each child. Serum testosterone, oestradiol and leptin were measured by specific radioimmunoassays, and HBA1c by high performance liquid chromatography.

RESULTS

Both diabetic girls and boys showed higher leptin levels than the normative healthy population and a group of age‐, sex‐ and BMI‐matched normal children. In an age‐related analysis, leptin levels in diabetic girls rose from 7.4 ± 1.2 and 8.1 ± 2.1 μg/l for the 5–7.99 and 8–10.99 year groups, to 12.6 ± 2.4 μg/l for the 11–13.99 year group, and to 15.6 ± 4.0 μg/l in the 14–15.99 year group in parallel with body weight. Leptin concentrations were parallel but higher (P < 0.05) than those of healthy girls. Diabetic boys had lower leptin levels than girls and, in contrast with normal boys, did not show a drop after the 10‐year period. Leptin levels were 4.9 ± 2.2, 3.9 ± 0.2, 5.5 ± 0.6 and 5.1 ± 0.9 μg/l for the 5–7.99, 8–10.99, 11–13.99 and 14–15.99 year groups, respectively. When divided by pubertal stage, leptin levels in the prepuberty stage of diabetic girls (8.6 ± 1.0 μg/l) were higher (P < 0.05) than those in the controls (4.1 ± 0.4 μg/l). In overt puberty girls, leptin was higher (P < 0.05) for diabetic (15.9 ± 2.9 μg/l) than for healthy girls (9.2 ± 1.1 μg/l). In prepubertal boys, differences were observed in leptin levels (4.9 ± 0.5 μg/l for diabetic boys and 3.4 ± 0.6 μg/l for healthy boys). In the overt puberty stage, diabetic boys showed higher (P < 0.05) levels of leptin (5.2 ± 0.7 μg/l) than the healthy matched controls (2.1 ± 0.2 μg/l). A multiple step regression analysis in the diabetic children revealed no associations between leptin and other relevant variables such as glycosylated haemoglobin, daily insulin dose, or years of suffering from the disease.

CONCLUSION

Serum leptin levels were higher in diabetic than in healthy children. These differences were not attributable to age, adiposity or stage of pubertal development, and were probably conditioned by the metabolic perturbation intrinsic to the diabetic state, or the chronic hyperinsulinemia.
The 1μg short Synacthen test in chronic fatigue syndrome
Authors: Hudson Marina , Cleare Anthony J.
Published: May 2003

OBJECTIVE

Many studies suggest mild hypocortisolism in chronic fatigue syndrome (CFS), usually assumed to be due to reduced suprahypothalamic drive to the hypothalamo‐pituitary‐adrenal (HPA) axis. We wished to explore further the state of the HPA axis in CFS using the 1 μg low dose short Synacthen test.

DESIGN

Subjects received an intravenous bolus of 1 μg Synacthen; samples for cortisol estimation were taken at baseline and 2, 10, 20, 30, 40 and 60 minutes after injection.

PATIENTS

We tested 20 subjects suffering from CFS according to the criteria of the Center for Diseases Control without psychiatric comorbidity and 20 matched healthy controls. All subjects were drug free for at least 1 month.

MEASUREMENTS

We calculated the cortisol responses to the test as the maximum cortisol attained, the incremental rise in cortisol over baseline (Δvalue) and as the integrated area under the curve.

RESULTS

There were no significant differences in baseline cortisol or cortisol responses between patients and controls. However, responses generally were low, and many subjects' peak responses were prior to the standard 30 minute sampling time., CONCLUSIONS These results do not lend support to the theory that patients with chronic fatigue syndrome have a low adrenal reserve. However, results from studies assessing the HPA axis are proving to be inconsistent. We suggest that many other factors may be contributing to HPA axis alterations in chronic fatigue syndrome, including sleep disturbance, inactivity, altered circadian rhythmicity, illness chronicity, concomitant medication and comorbid psychiatric disturbance. These sources of heterogeneity need to be considered in future studies, and may explain the inconsistent findings to date.
Patients' readiness for dietary change at the beginning of counselling: a transtheoretical model‐based assessment
Authors: Kasila K. , Poskiparta M. , Karhila P. , Kettunen T.
Published: May 2003

Background  The purpose of this study was to explore patients' readiness for dietary change within a theoretical framework of the transtheoretical model. The patients were recently diagnosed to have type 2 diabetes or impaired glucose tolerance. We discuss the theoretical aspects of appropriate dietary counselling strategies from a standpoint of patient‐specific stages of change.

Methods  The data included 32 audiotaped diabetes counselling sessions with 16 patients conducted by two nurses. The transcribed data was analysed by using deductive content analysis.

Results  The patients were at different stages of change of diabetes‐affected dietary behaviour. Their stages of change varied in different dietary areas and within certain dietary habits. These stages of change could involve their overall dietary behaviour or some minor aspects of their diets.

Conclusions  Understanding patient‐specific stages of change orientates health counsellors to use the most appropriate counselling strategies. The transtheoretical framework helps counsellors to perceive the total range of patients' different stages of change and their effect on the implementation of counselling. However, determining patients' stages of change through examining counselling conversations is occasionally difficult. Further qualitative research is called for.
Food provision and the nutritional implications of food choices made by young adult males, in a young offenders' institution
Authors: Eves A. , Gesch B.
Published: May 2003

The nutritional adequacy of diets provided by a prison was assessed by analysis of the kitchen menu for 1 week of a 4‐week cycle. Dietary intakes were determined using a predefined 7‐day diet diary in which prisoners indicated what they had eaten, and how much. A total of 159 prisoners took part in the study.

The food provided by the prison kitchen was broadly in line with current dietary recommendations. Vitamin content exceeded recommendations, with the exception of niacin in the vegetarian menu (12.6 mg compared with the reference nutrient intake of 16.8 mg). Selenium content was low in all menus, but particularly in the vegetarian menu in 1997 where it equalled the lower reference nutrient intake (LRNI) (39.5 μg). Food choices made by prisoners resulted in a wide variation in dietary intakes. Fat intake (as a proportion of energy) exceeded the recommended 35% in 82% of diets in 1996, and 64% of diets in 1997. In 1996, 34% of prisoners had intakes above 40% energy as fat. High fat intakes were largely the result of consuming items from the prison shop. Vitamin D intakes were low (3.4 and 3.3 μg in 1996 and 1997, respectively) compared with the recommendation (10 μg) for those with limited exposure to sunlight. Intakes of a number of minerals fell below recommendations, with some prisoners barely meeting the LRNI. This was particularly notable for selenium where 35% of prisoners in 1996, and 60% of prisoners in 1997 had intakes below the LRNI.
Sleep–wake rhythm in an irregular shift system
Authors: Sallinen Mikael , Härmä Mikko , Mutanen Pertti , Ranta Riikka , Virkkala Jussi , Müller Kiti
Published: May 2003

Sleep in shift work has been studied extensively in regular shift systems but to a lesser degree in irregular shifts. Our main aim was to examine the sleep–wake rhythm in shift combinations ending with the night or the morning shift in two irregular shift systems. Three weeks' sleep/work shift diary data, collected from 126 randomly selected train drivers and 104 traffic controllers, were used in statistical analyses including a linear mixed model and a generalized linear model for repeated measurements. The results showed that the sleep–wake rhythm was significantly affected by the shift combinations. The main sleep period before the first night shift shortened by about 2 h when the morning shift immediately preceded the night shift as compared with the combination containing at least 36 h of free time before the night shift (reference combination). The main sleep period before the night shift was most curtailed between two night shifts, on average by 2.9 and 3.5 h among the drivers and the controllers, respectively, as compared with the reference combination. Afternoon napping increased when the morning or the day shift immediately preceded the night shift, the odds being 4.35–4.84 in comparison with the reference combination. The main sleep period before the morning shift became 0.5 h shorter when the evening shift preceded the morning shift in comparison with the sleep period after a free day. The risk for dozing off during the shift was associated only with the shift length, increasing by 17 and 35% for each working hour in the morning and the night shift, respectively. The results demonstrate advantageous and disadvantageous shift combinations in relation to sleep and make it possible to improve the ergonomy of irregular shift systems.
Assessment of reproducibility of measures of intraocular pressure and central corneal thickness in young white adults over a 16‐h time period
Authors: Aakre Bente Monica , Doughty Michael J. , Dalane Olav V. , Berg Andreas , Aamodt Øystein , Gangstad Hedda
Published: May 2003

Purpose: The aims of this study were to obtain clinical measures of intraocular pressure (IOP) and central corneal thickness (CCT) and during waking hours to further assess both the nature of diurnal changes, and the reproducibility of any possible association between the two measures.

Methods: Fifteen white (Norwegian) healthy young adult subjects aged 20–29 years had IOP and CCT measures made, using a non‐contact specular microscope and non‐contact tonometry (NCT), every hour over a period of 16 h, starting at 06.30 hours. The experiment was repeated on two successive days.

Results: The average NCT readings were 12.5 and 11.4 mmHg for days 1 and 2, while pachometry averages were 0.518 and 0.514 mm. The range of NCT values, for any set of readings, averaged 2 mmHg, while this range was 0.014 mm for CCT measures. The reproducibility of these measures, as estimated by the coefficient of variation (COV) was 7.2% for NCT and 1.0% for pachometry measures. The COV for tonometry was inversely proportional to the actual values of these measures. A significant time‐related decline in tonometry values was seen over day 1 but not day 2. Pachometry values declined very rapidly over the first hour in both sessions and were essentially constant for the rest of the sessions. The strongest association between pachometry data and NCT measures was noted around midday (12.30 hours) on both sessions.

Conclusions: These studies confirm that the reproducibility (repeatability) of NCT and pachometry are substantially different. It is suggested that this aspect of tonometry needs to be taken into account, especially where there are lower values, when considering the possible impact of corneal thickness on tonometry measures and their interpretation. These types of effects need to be further assessed in older patients with ocular hypertension, or other glaucoma suspects.
Exploring New Gene Discoveries in Idiopathic Generalized Epilepsy
Authors: Noebels Jeffrey L.
Published: May 2003

Summary: Most epilepsies are categorized under the umbrella term “idiopathic;” these seizure disorders lack a known cause. New genetic technologies are rapidly identifying specific genes responsible for idiopathic generalized epilepsies (IGEs) and are gradually taking the “I” out of “IGE.” Ion channel (both voltage‐ and receptor‐mediated) mutations have been linked to a variety of epilepsies considered idiopathic. Gene errors alter excitability in various ways, depending on the mutation, the regional network, and the stage of brain development. The majority of mutations prolong depolarization, favor repetitive firing, and alter neurotransmitter release or postsynaptic sensitivity at central synapses, but the reason for specific seizure types is unclear. Further analyses of these gene mutations and their effects on the developing brain are providing critical clues in the search to explain the origin of “idiopathic” epilepsy.
ZP123 Increases Gap Junctional Conductance and Prevents Reentrant Ventricular Tachycardia During Myocardial Ischemia in Open Chest Dogs
Authors: Xing Dezhi , Kjølbye Anne Louise , Nielsen Morten S. , Petersen Jørgen S. , Harlow Kenneth W. , Holstein‐Rathlou Niels‐Henrik , Martins James B.
Published: May 2003

Introduction: The aim of this study was to determine if the stable antiarrhythmic peptide (AAP) analogue ZP123 increases gap junctional intercellular conductance and prevents reentrant ventricular tachycardia (VT) during coronary artery occlusion.

Methods and Results: Voltage clamp experiments demonstrated that 10 nM ZP123 improved gap junctional intercellular conductance by

69%± 20%

in pairs of guinea pig ventricular myocytes. VT was induced by programmed stimulation in α‐chloralose anaesthetized open chest dogs 1 to 4 hours after coronary artery occlusion. Three‐dimensional activation mapping was done using six bipolar electrograms on each of 23 multipolar needles in the risk zone. When VT was reproducibly induced, dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (intravenous bolus followed by 30‐min infusion per dose). Attempts to induce VT were repeated in each infusion period. Mass spectrometry was used to measure ZP123 plasma concentrations. Twenty‐six dogs with reentrant VT were included. ZP123 significantly prevented reentrant VT at all plasma concentrations vs saline:

1.0 ± 0.2 nM: 6/12 vs 0/12; 7.7 ± 0.6 nM: 7/13 vs 1/12

; and

69.2 ± 5.4 nM: 9/13 vs 1/13

. The preventive effect of ZP123 on reentrant VT was closely correlated to reversal of functional, unidirectional conduction block. ZP123 did not affect effective refractory period, surface ECG parameters, mean arterial pressure, or infarct size.

Conclusion: The stable AAP analogue ZP123 increased gap junctional intercellular conductance and specifically prevented the induction of reentrant VT during ischemia in a broad dose range without proarrhythmic or hemodynamic side effects. ZP123 is a promising candidate for use in preventing ischemia‐induced VT.
(J Cardiovasc Electrophysiol, Vol. 14, pp. 510‐520, May 2003)
Use of Electrogram Characteristics During Sinus Rhythm to Delineate the Endocardial Scar in a Porcine Model of Healed Myocardial Infarction
Authors: Wrobleski David , Houghtaling Christopher , Josephson Mark E. , Ruskin Jeremy N. , Reddy Vivek Y.
Published: May 2003

Introduction: Substrate‐based catheter ablation of postmyocardial infarction (post‐MI) ventricular tachycardia necessitates electroanatomic definition of the scarred endocardium. We sought to determine whether electrogram criteria during sinus rhythm could identify the location and extent of the myocardial scar by electroanatomic mapping.

Methods and Results: A porcine model of healed MI was generated by injecting agarose microspheres into the mid left anterior descending coronary artery. At least 4 weeks post‐MI, the animals

(n = 24)

underwent detailed left ventricular endocardial electroanatomic mapping using a 4‐mm‐tip catheter (BioSense‐Webster, Inc.). Based upon mapping data in normal animals, infarcted tissue was defined as bipolar electrogram amplitude <1.5 mV and electrogram duration ≥50 msec. Radiofrequency ablation lesions (2–10 per animal) were placed to tag the endocardial borders of the electroanatomic mapping‐defined scar. The area of the scar defined by abnormal voltage amplitude was

25.9 ± 15.4 cm2

(range 6.9–60.5). This area correlated well with that defined as scar by the electrogram duration criteria

(26.4 ± 16 cm2)

. Of those points remote from the infarct with falsely low voltage amplitude resulting from presumed poor catheter‐tissue contact, 94% were correctly identified as normal when using the electrogram duration criteria. Late potentials were observed predominantly along the borders of the infarcted myocardium. The radiofrequency lesions placed to tag the scar borders were located along the scar periphery during gross pathologic examination.

Conclusion: During normal sinus rhythm, both bipolar electrogram voltage amplitude and electrogram duration criteria are able to help differentiate normal from scarred myocardial tissue. Using these criteria, a detailed reconstruction of the endocardial scar can be rendered by electroanatomic mapping of the heart.
(J Cardiovasc Electrophysiol, Vol. 14, pp. 524‐529, May 2003)
Heart Rate Turbulence after Ventricular and Atrial Premature Beats in Subjects without Structural Heart Disease
Authors: Lindgren Kai S. , Mäkikallio Timo H. , Seppänen Tapio , Raatikainen M.J. Pekka , Castellanos Agustin , Myerburg Robert J. , Huikuri Heikki V.
Published: May 2003

Introduction: Studies assessing heart rate (HR) behavior after premature beats have focused on HR responses to ventricular premature beats (VBPs), but there is less information of HR behavior after atrial premature beats (APBs).

Methods and Results: HR turbulence after VPBs and APBs was first measured in response to ambient APBs and VPBs occurring during 24‐hour ambulatory ECG recordings in 29 subjects without structural heart disease, and in response to programmed atrial (AE) and ventricular extrastimuli (VE) in 6 subjects undergoing electrophysiologic (EP) examination. Turbulence onset (TO) was more negative

(−2.3 ± 3.2% vs −0.9 ± 2.8%, P < 0.01)

and turbulence slope (TS) was steeper

(11 ± 11 vs 5.1 ± 4.1 msec/R‐R interval, P < 0.05)

after VPBs than APBs. Compared to VPBs, the acceleration of HR after APBs was delayed by one beat, and APBs were associated with a short R‐R interval preceding the APB, resulting in a blunted TO. Studies of patients undergoing an EP test confirmed the one‐beat delay of HR acceleration and the blunted TO after programmed AE compared to VE

(P < 0.05)

. TO and TS after VPBs were related to baroreflex sensitivity. TO also was related to 24‐hour standard deviation of N‐N intervals (SDNN). However, the TO or TS following APBs was not related to either SDNN or baroreflex sensitivity.

Conclusion: HR behavior is different in response to APBs and VBPs among subjects without structural heart disease. Different definitions and calculation formulas should be used in the analysis of HR turbulence after APBs and VPBs.
(J Cardiovasc Electrophysiol, Vol. 14, pp. 447‐452, May 2003)
Modeling network dynamics
Authors: Vilar José M.G. , Guet Călin C. , Leibler Stanislas
Published: May 2003

We use the lac operon in Escherichia coli as a prototype system to illustrate the current state, applicability, and limitations of modeling the dynamics of cellular networks. We integrate three different levels of description (molecular, cellular, and that of cell population) into a single model, which seems to capture many experimental aspects of the system.
Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis
Authors: Berson Joanne F. , Theos Alexander C. , Harper Dawn C. , Tenza Danielle , Raposo Graça , Marks Michael S.
Published: May 2003

Lysosome-related organelles are cell type–specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell–specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.
Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments
Authors: Ackerley Steven , Thornhill Paul , Grierson Andrew J. , Brownlees Janet , Anderton Brian H. , Leigh P. Nigel , Shaw Christopher E. , Miller Christopher C.J.
Published: May 2003

Neurofilaments possess side arms that comprise the carboxy-terminal domains of neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates neurofilament transport. Analyses of neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows neurofilament transport, and this is due to increased pausing in neurofilament movement.
Structural Analysis of Human Rhinovirus Complexed with ICAM-1 Reveals the Dynamics of Receptor-Mediated Virus Uncoating
Authors: Xing Li , Casasnovas José M. , Cheng R. Holland
Published: May 2003

Intercellular adhesion molecule 1 (ICAM-1) functions as the cellular receptor for the major group of human rhinoviruses, being not only the target of viral attachment but also the mediator of viral uncoating. The configurations of HRV3-ICAM-1 complexes prepared both at 4°C and physiological temperature (37°C) were analyzed by cryoelectron microscopy and image reconstruction. The particle diameters of two complexes (with and without RNA) representing uncoating intermediates generated at 37°C were each 4% larger than that of those prepared at 4°C. The larger virus particle arose by an expansive movement of the capsid pentamers along the fivefold axis, which loosens interprotomer contacts, particularly at the canyon region where the ICAM-1 receptor bound. Particle expansion required receptor binding and preceded the egress of the viral RNA. These observations suggest that receptor-mediated uncoating could be a consequence of restrained capsid motion, where the bound receptors maintain the viral capsid in an expanded open state for subsequent genome release.
The Stress-Inducible Immediate-Early Responsive Gene IEX-1 Is Activated in Cells Infected with Herpes Simplex Virus 1, but Several Viral Mechanisms, Including 3′ Degradation of Its RNA, Preclude Expression of the Gene
Authors: Taddeo Brunella , Esclatine Audrey , Zhang Weiran , Roizman Bernard
Published: May 2003

The accumulation of cellular transcripts from cells infected with herpes simplex virus 1 (HSV-1) as measured with the aid of Affymetrix microchips has been reported elsewhere. Among these transcripts were genes that respond to stress and that could have a noxious effect on viral replication. We have selected the stress-inducible cellular gene encoding the immediate-early response protein IEX-1 to verify and determine the significance of the accumulation of these transcripts in infected cells. We report that we verified the increase in accumulation of IEX-1 transcripts after infection by Northern analyses and real-time PCR. These transcripts reach peak levels between 3 and 7 h after infection and decrease thereafter. However, IEX-1 protein was detected in cells 1 h after infection but not at later intervals. Studies designed to elucidate the failure of IEX-1 protein to be synthesized revealed the following points. (i) IEX-1 RNA transported to the cytoplasm after 1 h of infection consisted of at least two populations, a partially degraded population and a population consisting of unspliced IEX-1 RNA. Neither of these RNAs could translate the authentic IEX-1 protein. (ii) The partially degraded IEX-1 RNA was not detected in the cytoplasm of cells infected with a mutant virus lacking the U
L
41 gene encoding the virion host shutoff protein (
vhs
). Although degradation of RNA mediated by
vhs
was reported to be 5′ to 3′, the partially degraded IEX-1 RNA lacked the 3′ sequences rather than the 5′ sequences. (iii) The unspliced pre-RNA form containing the IEX-1 intron sequences was detected in the cytoplasm of cell infected with wild-type virus but not in those infected with a mutant lacking the α27 gene encoding the infected cell protein No. 27. (iv) Overexpression of IEX-1 protein by transduction of the gene prior to infection with 1 PFU of HSV-1 per cell had no effect on the accumulation of late genes and virus yield. We conclude that the failure of IEX-1 to express its protein reflects the numerous mechanisms by which the virus thwarts the cells from expressing its genes after infection.
CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression
Authors: Badal Vinay , Chuang Linda S. H. , Tan Eileen Hwee-Hong , Badal Sushma , Villa Luisa L. , Wheeler Cosette M. , Li Benjamin F. L. , Bernard Hans-Ulrich
Published: May 2003

Infection with genital human papillomaviruses (HPVs) is the primary cause of cervical cancer. The infection is widespread, and little is known about the secondary factors associated with progression from subclinical infection to invasive carcinoma. Here we report that HPV genomes are efficiently targeted in vivo by CpG methylation, a well-known mechanism of transcriptional repression. Indeed, it has been shown previously that in vitro-methylated HPV type 16 (HPV-16) DNA is transcriptionally repressed after transfection into cell cultures. By using a scan with the restriction enzyme McrBC, we observed a conserved profile of CpG hyper- and hypomethylation throughout the HPV-16 genomes of the tumor-derived cell lines SiHa and CaSki. Methylation is particularly high in genomic segments overlying the late genes, while the long control region (LCR) and the oncogenes are unmethylated in the single HPV-16 copy in SiHa cells. In 81 patients from two different cohorts, the LCR and the E6 gene of HPV-16 DNA were found to be hypermethylated in 52% of asymptomatic smears, 21.7% of precursor lesions, and 6.1% of invasive carcinomas. This suggests that neoplastic transformation may be suppressed by CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression. These prevalences of hyper- and hypomethylation also indicate that CpG methylation plays an important role in the papillomavirus life cycle, which takes place in asymptomatic infections and precursor lesions but not in carcinomas. Bisulfite modification revealed that in most of the HPV-16 genomes of CaSki cells and of asymptomatic patients, all 11 CpG dinucleotides that overlap with the enhancer and the promoter were methylated, while in SiHa cells and cervical lesions, the same 11 or a subset of CpGs remained unmethylated. Our report introduces papillomaviruses as models to study the mechanism of CpG methylation, opens research on the importance of this mechanism during the viral life cycle, and provides a marker relevant for the etiology and diagnosis of cervical cancer.
Palmitoylation of the
Autographa californica
Multicapsid Nucleopolyhedrovirus Envelope Glycoprotein GP64: Mapping, Functional Studies, and Lipid Rafts
Authors: Zhang Sandy Xiaoxin , Han Yu , Blissard Gary W.
Published: May 2003

Budded virions (BV) of the baculovirus
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in Ac
M
NPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type Ac
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NPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) Ac
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NPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.
Hepatitis B Virus X Protein and Simian Virus 5 V Protein Exhibit Similar UV-DDB1 Binding Properties To Mediate Distinct Activities
Authors: Leupin Olivier , Bontron Séverine , Strubin Michel
Published: May 2003

The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.
Transport of the Intracisternal A-Type Particle Gag Polyprotein to the Endoplasmic Reticulum Is Mediated by the Signal Recognition Particle
Authors: Fehrmann Frauke , Jung Martin , Zimmermann Richard , Kräusslich Hans-Georg
Published: May 2003

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.
Transformation of Rodent Fibroblasts by the Jaagsiekte Sheep Retrovirus Envelope Is Receptor Independent and Does Not Require the Surface Domain
Authors: Chow Yen-Hung J. , Alberti Alberto , Mura Manuela , Pretto Carla , Murcia Pablo , Albritton Lorraine M. , Palmarini Massimo
Published: May 2003

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). Expression of the JSRV envelope protein (Env) is sufficient to transform immortalized and primary fibroblasts, but the precise mechanisms of this process are not known. The cellular receptor for JSRV is hyaluronidase 2 (Hyal-2), the product of a putative tumor suppressor gene that in humans maps to a chromosomal region frequently deleted in the development of lung and breast cancers. Here we report studies to determine whether the Hyal-2-JSRV Env interaction plays a role in virus-induced transformation of rodent fibroblasts. Chimeric Env proteins between JSRV and the unrelated murine retroviruses Moloney murine leukemia virus (MMuLV) and mouse mammary tumor virus (MMTV) showed cell surface expression comparable to that of wild-type MMuLV Env and rescued infection of MMuLV particle pseudotypes. Interestingly, an MMuLV-JSRV chimera in which the putative receptor binding domain (RBD) and proline-rich region (PRR) of JSRV Env were replaced by the RBD and PRR of MMuLV induced transformation of 208F, a rodent fibroblast line. Cell lines derived from foci of MMuLV-JSRV chimera-transformed 208F cells grew in soft agar and showed Akt activation, a hallmark of JSRV-transformed rodent fibroblasts. Transformation assays performed using proteins with amino-terminal deletion mutations showed that the carboxy-terminal 141 amino acids of the transmembrane subunit (TM) were sufficient to induce cell transformation when targeted to the membrane with a myristoylation signal. Thus, the JSRV TM is necessary and sufficient to transform rodent fibroblasts. Taken together these results indicate that the interaction with Hyal-2 at least is not an essential determinant of JSRV-induced transformation of fibroblasts and that the viral TM functions essentially as an oncoprotein.
U.S. Human Immunodeficiency Virus Type 1 Epidemic: Date of Origin, Population History, and Characterization of Early Strains
Authors: Robbins Kenneth E. , Lemey Philippe , Pybus Oliver G. , Jaffe Harold W. , Youngpairoj Ae S. , Brown Teresa M. , Salemi Marco , Vandamme Anne-Mieke , Kalish Marcia L.
Published: May 2003

Human immunodeficiency virus (HIV) type 1 subtype B sequences (whole envelope and the p17 region ofgag) were obtained from peripheral blood mononuclear cell samples collected in 1981 from seven HIV-infected U.S. individuals and in 1982 from one infected Canadian resident. Phylogenetic and nucleotide distance analyses were performed by using database sequences representing North American strains collected from 1978 to 1995. The estimated phylogeny was starlike, with early strains represented on different lineages. When sequences were grouped by years of collection, nucleotide distance comparisons demonstrated an increase in diversity over time and indicated that contemporary strains are more closely related to early epidemic strains than to each other. Using a recently developed likelihood ratio reduction procedure, the date of origin of the U.S. epidemic was estimated to be 1968 ± 1.4 years. A coalescent approach was also used to estimate the population history of the U.S. subtype B epidemic. Our analyses provide new information that implies an exponential growth rate from the beginning of the U.S. HIV epidemic. The dating results suggest a U.S. introduction date (or date of divergence from the most recent common ancestor) that precedes the date of the earliest known AIDS cases in the late 1970s. Furthermore, the estimated epidemic growth curve shows a period of exponential growth that preceded most of the early documented cases and also indicates a leveling of prevalence rates in the recent past.
Intracellular Targeting of Gag Proteins of theDrosophilaTelomeric Retrotransposons
Authors: Rashkova S. , Athanasiadis A. , Pardue M.-L.
Published: May 2003

Drosophilahas two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements,HeT-AandTART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites,HeT-AandTARTtranspose only to chromosome ends. AlthoughHeT-AandTARTbelong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei whereHeT-AGag forms structures associated with telomeres.TARTGag does not associate with telomeres unlessHeT-AGag is present, suggesting a symbiotic relationship in whichHeT-AGag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in culturedDrosophilacells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only inHeT-AGag. These are present at several places in the protein, are not redundant, and cannot be complemented intrans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.
Unique Pattern of Convergent Envelope Evolution in Simian Immunodeficiency Virus-Infected Rapid Progressor Macaques: Association with CD4-Independent Usage of CCR5
Authors: Dehghani Houman , Puffer Bridget A. , Doms Robert W. , Hirsch Vanessa M.
Published: May 2003

The rate of disease development in simian immunodeficiency virus (SIV) infection of macaques varies considerably among individual macaques. While the majority of macaques inoculated with pathogenic SIV develop AIDS within a period of 1 to 2 years, a minority exhibit a rapid disease course characterized by absence or transience of humoral and cellular immune responses and high levels of virus replication with widespread dissemination of SIV in macrophages and multinucleated giant cells. The goal of this study was to examine viral evolution in three SIVsmE543-3-inoculated rapid progressors to determine the contribution of viral evolution to the development of rapid disease and the effect of the absence of immune pressure upon viral evolution. PCR was used to amplify and clone the entire SIV genome from tissues collected at necropsy, and the course of viral evolution was assessed byenvsequences cloned from sequential plasma samples of one rapid progressor (RP) macaque. The majority of sequence changes in RP macaques occurred in the envelope gene. Substitutions were observed in all three animals at specific conserved residues in envelope, including loss of a glycosylation site in V1/V2, a D-to-N/V substitution in a highly conserved GDPE motif, and a P-to-V/H/T substitution in the V3 loop analog. A cell-cell fusion assay revealed that representativeenvclones utilized CCR5 as a coreceptor, independent of CD4. The selection of specific substitutions in envelope in RP macaques suggests novel selection pressures on virus in such animals and suggests that viral variants that evolve in these animals may play a role in disease progression.
Characterization of a Conformational Epitope on Hepatitis B Virus Core Antigen and Quasiequivalent Variations in Antibody Binding
Authors: Conway J. F. , Watts N. R. , Belnap D. M. , Cheng N. , Stahl S. J. , Wingfield P. T. , Steven A. C.
Published: May 2003

We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to ∼10-Å resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides—two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid—T=3 and T=4 particles—present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from ∼100 to ∼0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.