Vitamin B12 and hepatitis C: Molecular biology and human pathology

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the H(IV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV), We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host), The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

Familial corticosteroid-binding globulin deficiency due to a noval null mutation: Association with fatigue and relative hypotension

Corticosteroid-binding globulin is a 383-amino acid glycoprotein that serves a hormone transport role and may have functions related to the stress response and inflammation. We describe a 39-member Italian-Australian family with a novel complete loss of function (null) mutation of the corticosteroid-binding globulin gene. A second, previously described, mutation (Lyon) segregated independently in the same kindred. The novel exon 2 mutation led to a premature termination codon corresponding to residue -12 of the procorticosteroid-binding globulin molecule (c.121G->A). Among 32 family members there were 3 null homozygotes, 19 null heterozygotes, 2 compound heterozygotes, 3 Lyon heterozygotes, and 5 individuals without corticosteroid-binding globulin mutations. Plasma immunoreactive corticosteroid-binding globulin was undetectable in null homozygotes, and mean corticosteroid-binding globulin levels were reduced by approximately 50% at 18.7 ± 1.3 µg/ml (reference range, 30–52 µg/ml) in null heterozygotes. Morning total plasma cortisol levels were less than 1.8 µg/dl in homozygotes and were positively correlated to the plasma corticosteroid-binding globulin level in heterozygotes. Homozygotes and heterozygote null mutation subjects had a high prevalence of hypotension and fatigue. Among 19 adults with the null mutation, the systolic blood pressure z-score was 12.1 ± 3.5; 11 of 19 subjects (54%) had a systolic blood pressure below the third percentile. The mean diastolic blood pressure z-score was 18.1 ± 3.4; 8 of 19 subjects (42%) had a diastolic blood pressure z-score below 10. Idiopathic chronic fatigue was present in 12 of 14 adult null heterozygote subjects (86%) and in 2 of 3 null homozygotes. Five cases met the Centers for Disease Control criteria for chronic fatigue syndrome. Fatigue questionnaires revealed scores of 25.1 ± 2.5 in 18 adults with the mutation vs. 4.2 ± 1.5 in 23 healthy controls (P < 0.0001). Compound heterozygosity for both mutations resulted in plasma cortisol levels comparable to those in null homozygotes. Abnormal corticosteroid-binding globulin concentrations or binding affinity may lead to the misdiagnosis of isolated ACTH deficiency. The mechanism of the association between fatigue and relative hypotension is not established by these studies. As idiopathic fatigue disorders are associated with relatively low plasma cortisol, abnormalities of corticosteroid-binding globulin may be pathogenic.

A field trial of the effect of improved piglet management on smallholder sow productivity in the Philippines

We conducted a randomised, controlled field trial during 1998/1999 to evaluate the hypothesis that improved piglet management would improve the reproductive performance of smallholder sows. Simple changes were introduced into the treatment herds including the construction of a heated piglet-separation pen, vitamin injections, creep feeding and early weaning. The control herds were unchanged. Data were collected from all sows in each enrolled herd over two farrowings. We enrolled 176 sows, including 170 (96 treatment and 74 control) sows that remained throughout the study period. Significant differences in the reproductive performance of treatment and control sows were recorded for interfarrowing interval (median 176 versus 220 days), average number liveborn over 2 litters (11 versus 12), and average preweaning mortality over 2 litters (0 versus 37%). Based on a discount rate of 17%, the benefit-cost ratio of the treatment was 11.1 and 12.1 over 3 and 5 years, respectively. (C) 2001 Elsevier Science B.V. All rights reserved.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Vitamin B12 stalls the 80 S ribosomal complex on the hepatitis C internal ribosome entry site

The effect of cyanocobalamin (CNCbl, vitamin 1312) on hepatitis C virus internal ribosome entry site (HCV IRES)-dependent initiation of translation was studied by ribosomal toeprinting and sucrose gradient centrifugation analysis. These results suggested that CNCbl did not inhibit HCV IRES-dependent translation by a competitive binding mechanism. CNCbl allowed 80 S elongation complex formation on the mRNA, but stalled the initiation at that point, effectively trapping the 80 S ribosomal complexes on the HCV TRES. CNCbl had no effect on cap-dependent mRNA, consistent with the known mRNA specificity of this translational inhibitor. To help elucidate the mechanism, comparative data were collected for the well-characterised translation inhibitors cycloheximide and 5'-guanylyl-imidophosphate, Although CNCbl stalled HCV IRES-dependent translation at approximately the same step in initiation as cycloheximide, the mechanisms of these two inhibitors are distinct. (C) 2002 Elsevier Science Ltd. All rights reserved.

Interval training program optimization in highly trained endurance cyclists

Purpose: The purpose of this study was to examine the influence of three different high-intensity interval training (HIT) regimens on endurance performance in highly trained endurance athletes. Methods: Before, and after 2 and 4 wk of training, 38 cyclists and triathletes (mean +/- SD; age = 25 +/- 6 yr; mass = 75 +/- 7 kg; (V)over dot O-2peak = 64.5 +/- 5.2 mL.kg(-1).min(-1)) performed: 1) a progressive cycle test to measure peak oxygen consumption ((V)over dotO(2peak)) and peak aerobic power output (PPO), 2) a time to exhaustion test (T-max) at their (V)over dotO(2peak) power output (P-max), as well as 3) a 40-kin time-trial (TT40). Subjects were matched and assigned to one of four training groups (G(1), N = 8, 8 X 60% T-max P-max, 1:2 work:recovery ratio; G(2), N = 9, 8 X 60% T-max at P-max, recovery at 65% HRmax; G(3), N = 10, 12 X 30 s at 175% PPO, 4.5-min recovery; G(CON), N = 11). In addition to G(1) G(2), and G(3) performing HIT twice per week, all athletes maintained their regular low-intensity training throughout the experimental period. Results: All HIT groups improved TT40 performance (+4.4 to +5.8%) and PPO (+3.0 to +6.2%) significantly more than G(CON) (-0.9 to + 1.1 %; P < 0.05). Furthermore, G(1) (+5.4%) and G(2) (+8.1%) improved their (V)over dot O-2peak significantly more than G(CON) (+ 1.0%; P < 0.05). Conclusion: The present study has shown that when HIT incorporates P-max as the interval intensity and 60% of T-max as the interval duration, already highly trained cyclists can significantly improve their 40-km time trial performance. Moreover, the present data confirm prior research, in that repeated supramaximal HIT can significantly improve 40-km time trial performance.

Identification of micronutrient deficiency of wheat in the Peshawar Valley, Pakistan

A field experiment was conducted to study the effect of micronutrients, zinc (Zn), copper (Cu), iron (Fe), manganese (Mn), boron (13) and a commercial fritted micronutrient product called Zarzameen, on the yield and the yield components of wheat (Triticum aestivum L.), in the Peshawar valley, Pakistan. Different combinations of Zn, Cu. Fe. Mn, B, and Zarzameen were applied at the rate of 4.0, 2.0, 5.0, 2.0, 1.0 kg ha(-1) and 1.0 kg ha(-1), respectively, along with a basal dose of 100 kg ha(-1) nitrogen(N), 75 kg ha(-1) phosphorus (P) and 50 kg ha(-1) potassium (K). The fertilizer treatments (macro- and micronutrients) increased wheat dry matter, grain yield, and straw yield significantly over an unfertilized control. Soil tests for B and Zn were increased both at boot and harvesting stage, and Fe at boot stage, with the addition of micronutrients. Plants without B had showed classical B deficiency symptoms at grain formation stage, but not at vegetative stage. Boron concentration in the dry matter of wheat plants increased with the addition of the B fertilizer in the soil. Boron deficiency was not observed in plants containing >4 mg B kg(-1) at the boot stage, or in soils containing > 1.4 mg kg(-1) hot water soluble B.

Sertoli-Leydig Cell Tumor of the Ovary, a Rare Cause of Precocious Puberty in a 12-Month-Old Infant

We report a 12-month-old infant who presented with a 4-month history of isosexual precocious puberty secondary to an estrogenizing Sertoli-Leydig cell tumor of the ovary. Total serum immunoreactive inhibin and subunits A and B were markedly elevated before surgical resection and subsequently decreased 7 wk later into the normal prepubertal range. Twenty weeks following surgical removal, the patient presented again with central precocious puberty; inhibin B levels were raised on this occasion, a luteinizing releasing hormone stimulation test confirmed central precocious puberty. This is the youngest reported occurrence of this rare sex cord stromal neoplasm. The prognosis of this extremely rare tumor presenting at this early juvenile stage is uncertain. This report illustrates the usefulness of serum inhibin as a tumor marker during therapeutic suppression with leuprorelin acetate for central precocious puberty. Analysis of genomic and tumor DNA revealed a normal nucleotide sequence for the LH receptor and the G{alpha}s gene. To understand the molecular pathogenesis of this tumor we analyzed mRNA levels for the inhibin A and B subunits, FSH receptor, LH receptor aromatase, steroidogenic factor-1 and the ER ß genes. Molecular characterization reveals the presence of genes specific for granulosa and Leydig cells; the relative expression of these genes, in addition to its histologic characteristics, suggests that this tumor may result from a dysdifferentiation of a primordial follicle.

Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH), a mutant that causes autosomal dominant GH deficiency type II

Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wildtype and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mm BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wildtype hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release.

Usefulness of B-type natriuretic peptide in hypertensive patients with exertional dyspnea and normal left ventricular ejection fraction and correlation with new echocardiographic indexes of systolic and diastolic function

B-type natriuretic peptide (BNP) levels increase in systolic heart failure (HF). However, the value of BNP in hypertensive patients with suspected diastolic HF (symptoms suggestive of HF but normal ejection fraction) and its relation to myocardial function in these patients is unclear. We prospectively studied 72 ambulatory hypertensive subjects (40 women, mean age 58 +/- 8 years) with exertional dyspnea and ejection fraction greater than or equal to50%. Diastolic function was evaluated with transmitral and pulmonary venous Doppler, mitral annular velocities (pulsed-wave tissue Doppler), and flow propagation velocity (color M-mode). Systolic function was assessed with strain and strain rate derived from color tissue Doppler imaging. BNP was related to myocardial function and the presence or absence of global diastolic dysfunction. By conventional Doppler criteria, 34 patients had normal left ventricular diastolic function and 38 had isolated diastolic dysfunction. BNP values were higher in patients with diastolic dysfunction (46 +/- 48 vs 20 +/- 20 pg/ml, p = 0.004) and were related independently to blood pressure, systolic strain rate, left atrial function (p < 0.01 for all), and age (p = 0.015). Patients with diastolic dysfunction and pseudonormal filling had higher BNP levels compared with impaired relaxation (89 +/- 47 vs 35 +/- 42 pg/ml, p = 0.001). However, 79% of patients with diastolic dysfunction had BNP levels within the normal range. We conclude that in ambulatory hypertensive patients with symptoms suggestive of mild HF and normal ejection fraction, BNP is related to atrial and ventricular systolic parameters, blood pressure, and age. Although elevated in the presence of diastolic dysfunction, the BNP level mostly is in the normal range and, therefore, has limited diagnostic value in stable patients with suspected diastolic HF. (C) 2003 by Excerpta Medica, Inc.

Abnormalities of the PTH-vitamin D axis and bone turnover markers in children, adolescents and adults with cystic fibrosis: comparison with healthy controls

Abnormalities of calcium and vitamin D metabolism in cystic fibrosis (CF) are well documented. We tested the hypothesis that alterations in calcium metabolism are related to vitamin D deficiency, and that bone resorption is increased relative to accretion in patients with CF. Calcitropic hormones, electrolytes, osteocalcin (OC) and bone alkaline phosphatase (BAP), (markers of bone mineralisation), urinary deoxypyridinoline [total (t) Dpd, a marker of bone resorption] and lumbar spine bone mineral density (LS BMD), expressed as a z-score, were measured in 149 (81 M) CF and 141 (61 M) control children aged 5.3-10.99 years, adolescents aged 11-17.99 years and adults aged 18-55.9 years. Data were analysed by multiple regression to adjust for age. In patients, FEV1% predicted and CRP (as disease severity markers), genotype and pancreatic status (PS) were recorded. The distribution of PTH differed between groups (P