ISOLATION AND CHARACTERIZATION OF THE CYANOGEN BROMIDE FRAGMENTS OF LOBSTER ARGININE KINASE (HOMARUS VULGARIS)

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10%) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to different gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position; therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs

The Mycobacterium tuberculosis phoP mutant strain SO2 has previously been shown to have reduced multiplication in mouse macrophages and in vivo using the mouse intravenous-infection model. In this study we demonstrate that the M. tuberculosis SO2 is highly attenuated when compared with the parental M. tuberculosis MT103 strain and also more attenuated than BCG in severe combined immunodeficiency disease (SCID) mice. Complementation of the M. tuberculosis SO2 with the wild-type phoP gene restored the virulence of the strain in the SCID mice, confirming that the attenuated phenotype is due to the phoP mutation. In Balb/c mice subcutaneously vaccinated with either M. tuberculosis SO2 or BCG, the proportions of CD4+ and CD8+ populations measured in the spleen were significantly higher in the M. tuberculosis SO2 vaccinated group. In addition, the proportion of antigen-stimulated CD4+/CD8+ cells expressing IFN-gamma was significantly higher in the M. tuberculosis SO2 vaccinated group when compared with the BCG group. Balb/c mice subcutaneously vaccinated with the M. tuberculosis SO2 strain were also protected against intra-venous challenge with M. tuberculosis H37Rv at levels comparable to mice vaccinated with BCG, as measured by reduced bacterial counts in lung and spleens. Guinea pigs subcutaneously vaccinated with the M. tuberculosis SO2 strain were protected against aerosol challenge with M. tuberculosis H37Rv delivered at different doses. A high dose aerosol challenge of M. tuberculosis SO2 vaccinated guinea pigs resulted in superior levels of protection when compared with BCG vaccination, as measured by guinea pig survival and reduction in disease severity in the lung.     

H244R VSX1 is associated with selective cone ON bipolar cell dysfunction and macular degeneration in a PPCD family

PURPOSE: To elucidate the retinal dysfunction and the molecular basis of posterior polymorphous corneal dystrophy (PPCD) associated with macular dystrophy, both inherited in a dominant manner through a three-generation family. METHODS: Ophthalmologic examinations including slit lamp examination, visual acuity tests, fundus visualization by scanning laser ophthalmoscopy, fluorescein angiography, color vision tests, electro-oculography, photopic and scotopic electroretinography (ERG) according to the International Society for Clinical Electrophysiology of Vision (ISCEV) protocols, and oscillatory potential (OP) recordings were conducted on affected family members. Corneal button from one affected patient was examined by transmission electron microscopy. All exons and intron-exon boundaries of the VSX1 and the COL8A2 genes were amplified by polymerase chain reaction and sequenced. RESULTS: The presence of endothelial cells that have epithelial-like features with multiple layers, desmosomal junctions, and microvillous projections supports the diagnosis of PPCD. Sequence analysis indicated that the H244R variant in the VSX1 segregated with corneal and macular disease phenotypes in this family. Electrophysiologic studies indicated normal scotopic ERG findings, decreased amplitude of the photopic b-wave, photopic OP2 and OP3 barely recordable with a preserved OP4 amplitude, and variably decreased 30-Hz flicker amplitude. CONCLUSIONS: The human VSX1 is required for cone ON bipolar cell function but not for rod and cone OFF bipolar cells, giving a unique example of such a selective heritable retinal defect in humans. Furthermore, the authors provide the first clinical support for a new alternative role of VSX1 in cone biology, probably similar to that proposed for its goldfish ortholog during retinal differentiation.     

Breast carcinoma during pregnancy. International recommendations from an expert meeting

BACKGROUND: Breast carcinoma during pregnancy (BCP) is a difficult clinical situation, as it appears to put the health of the mother in conflict with that of the fetus. METHODS: An international expert meeting was conducted to form guidelines on how to diagnose and treat women with BCP. RESULTS: The goal for treatment of the pregnant woman with breast carcinoma is the same as that of the nonpregnant breast carcinoma patient: local control of disease and prevention of systemic metastases. However, certain treatment modalities need to be modified because of the potential for adverse effects on the fetus. There is evidence to support the safety of anthracycline-based chemotherapy during the second and third trimesters of pregnancy (Oxford Level of Evidence [LOE] 2b). Because of the lack of evidence, the expert opinion was not to recommend the routine use of newer cytotoxic drugs like the taxanes during pregnancy (LOE 5). CONCLUSION: The recommendations provided should help to reach informed decision making by the patient. The ongoing prospective collection of data on BCP, such as that at the University of Texas M.D. Anderson Cancer Center (UTMDACC) and that of the German Breast Group/Breast International Group (GBG/BIG), is necessary to further our knowledge regarding the treatment of this unique group of breast carcinoma patients.     

High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112_L113insR, 92% of family's mutations) and the Netherlands (c.225_243del19, 90% of family's mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available.     

Community surveys and risk factor analysis of human alveolar and cystic echinococcosis in Ningxia Hui Autonomous Region, China

OBJECTIVE: To determine the true community prevalence of human cystic (CE) and alveolar (AE) echinococcosis (hydatid disease) in a highly endemic region in Ningxia Hui, China, by detecting asymptomatic cases. METHODS: Using hospital records and "AE-risk" landscape patterns we selected study communities predicted to be at risk of human echinococcosis in Guyuan, Longde and Xiji counties. We conducted community surveys of 4773 individuals from 26 villages in 2002 and 2003 using questionnaire analysis, ultrasound examination and serology. FINDINGS: Ultrasound and serology showed a range of prevalences for AE (0-8.1%; mean 2%) and CE (0-7.4%; mean 1.6%), with the highest prevalence in Xiji (2% for CE, 2.5% for AE). There were significant differences in the prevalence of CE, AE and total echinococcosis between the three counties and villages (with multiple degrees of freedom). While hospital records showed 96% of echinococcosis cases attributable to CE, our survey showed a higher prevalence of human AE (56%) compared to CE (44%). Questionnaire analysis revealed that key risk factors for infection were age and dog ownership for both CE and AE, and Hui ethnicity and being female for AE. Drinking well-water decreased the risk for both AE and CE. CONCLUSION: Echinococcosis continues to be a severe public health problem in this part of China because of unhygienic practices/habits and poor knowledge among the communities regarding this disease.     

Prognosis of lupus membranous nephropathy in children

The occurrence of membranous nephropathy in pediatric series of systemic lupus erythematosus has been reported only rarely, probably due to a very low frequency. One hundred fifty-four children who were seen in 100 French pediatric centers between January 2002 and April 2005 were included. Fifteen (12 girls and three boys) out of the 81 (18.5 %) children with renal involvement presented histological features of membranous nephropathy. Their ages ranged from six to 15 years old (mean=11.3) at the age of SLE diagnosis and 8/15 children were of African origin. Isolated membranous nephropathy was observed in nine patients, of whom five patients displayed a complete recovery following immunosuppressive treatment. Associated proliferative lesions were observed on the first kidney specimen in two patients and in a further renal biopsy in four other patients, leading to a less favorable course of lupus nephropathy.     

Chloroacetaldehyde- and acrolein-induced death of human proximal tubule cells

Ifosfamide (ifo) is a commonly used drug in chemotherapy. It is metabolized to acrolein (acro) and chloroacetaldehyde (CAA), which are thought to be responsible for renal side effects. We studied the effects of ifo and cyclophosphamide (cyclo) as well as their metabolites, acro and CAA, on cellular protein content, necrosis, apoptosis and cytosolic calcium concentration using a human proximal tubule cell line. The protein content decreased during acro or CAA administration (15 to 300 µmol/l), but not during ifo or cyclo exposure over a time period of up to 72 h. Mild apoptosis was induced only by high acro (150, 300 µmol/l) and low CAA concentrations (15, 75 µmol/l) and only in a narrow time window (24 h). Necrosis was increased after exposure to acro or CAA at all concentrations. CAA was more potent than acro. Ifo and cyclo did not induce necrosis or apoptosis. Glutathione abolished CAA-induced cell death. Cytosolic calcium concentrations increased after acro or CAA administration and showed an oscillating pattern. Cytosolic Ca²⁺ chelation did not prevent necrosis. We conclude that neither ifo nor cyclo induce cell damage, but that their metabolites acro and CAA induce cell death. This cell death occurs mainly by necrosis and not by apoptosis.M. Gekle and N. Gordjani contributed equally to this work     

Systemic nitric oxide augmentation leads to a rapid decrease of the bladder outlet resistance in healthy men

OBJECTIVE: We examined the immediate effect of a systemic nitric oxide augmentation on the bladder outlet resistance in healthy men. METHODS: Eleven healthy male volunteers with a mean age of 25.5 yr were included in the study. They were prepared for a standard urodynamic study, and a baseline pressure-flow study was obtained. The subjects were then given 20 mg isosorbide dinitrate sublingually, and after refilling their bladder a second pressure-flow study was done after 20 min. The pressure-flow studies were then compared in regard to the average flow rate, the average detrusor pressure during micturition, and the detrusor pressure at maximum flow rate. RESULTS: One of the subjects was unable to void and had to be excluded from the study. In the remaining 10 men, the mean average flow rate increased from 16.7 ml/s before to 20.2 ml/s after the intake of the NO donor (P=0.013). Concomitantly, the average detrusor pressure during micturition decreased from a mean of 57 to 52 cm H2O (P=0.004) and the mean detrusor pressure at maximum flow rate decreased from 60 to 52 cm H2O (P=0.013). CONCLUSIONS: Systemic NO augmentation can lower the functional bladder outlet resistance very rapidly in men. Our results support the concept that the NO-cGMP pathway may be a promising target for medical treatment of lower urinary tract symptoms.