Cushing disease due to a somatic USP8 mutation in a patient with evolving pituitary hormone deficiencies due to a germline GH1 splicing variant

SUMMARY We present the unique case of an adult Brazilian woman with severe short stature due to growth hormone deficiency with a heterozygous G to T substitution in the donor splice site of intron 3 of the growth hormone 1 (GH1) gene (c.291+1G>T). In this autosomal dominant form of growth hormone deficiency (type II), exon 3 skipping results in expression of the 17.5 kDa isoform of growth hormone, which has a dominant negative effect over the bioactive isoform, is retained in the endoplasmic reticulum, disrupts the Golgi apparatus, and impairs the secretion of other pituitary hormones in addition to growth hormone deficiency. This mechanism led to the progression of central hypothyroidism in the same patient. After 5 years of growth and thyroid hormone replacement, at the age of 33, laboratory evaluation for increased weight gain revealed high serum and urine cortisol concentrations, which could not be suppressed with dexamethasone. Magnetic resonance imaging of the sella turcica detected a pituitary macroadenoma, which was surgically removed. Histological examination confirmed an adrenocorticotropic hormone (ACTH)-secreting pituitary macroadenoma. A ubiquitin-specific peptidase 8 (USP8) somatic pathogenic variant (c.2159C>G/p.Pro720Arg) was found in the tumor. In conclusion, we report progression of isolated growth hormone deficiency due to a germline GH1 variant to combined pituitary hormone deficiency followed by hypercortisolism due to an ACTH-secreting macroadenoma with a somatic variant in USP8 in the same patient. Genetic studies allowed etiologic diagnosis and prognosis of this unique case.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Superoxide overproduction and kidney fibrosis: a new animal model / Produção aumentada de superóxido e fibrose renal: novo modelo animal

Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. .

Objetivo Estabelecer se a mutação no gene Immp2L induz à fibrose renal e se o envelhecimento exacerba a morfologia renal em camundongos. Métodos Foram usadas fêmeas de camundongos mutantes para proteína semelhante à peptidase 2 da camada interna da mitocôndria, com 3 e 18 meses de idade. Para analisar a fibrose renal, foram usados o escore clássico de fibrose, a coloração com tricrômio de Masson, e a análise de marcadores profibróticos, por meio da reação em cadeia de polimerase em tempo real (superóxido dismutase 1, metalonoproteinase-9, eritropoietina e fator transformador de crescimento beta), e a imunocoloração (fibroblastos e colágeno IV). Marcadores de estresse oxidativo foram determinados por imuno-histoquímica. O número de células apoptóticas renais foi analisado. A função renal foi estimada por creatinina sérica. Resultados Camundongos mutantes jovens apresentaram glomeruloesclerose em quantidade significativamente maior que animais da mesma idade (p=0,034). Os mutantes mostraram maior formação de cilindros tubulares (p=0,025), deposição de colágeno (p=0,019) e maior expressão de colágeno do tipo IV (p<0,001). A expressão de superóxido dismutase 1 foi maior em mutantes jovens (p=0,038). Mutantes idosas exibiram maior expressão dos marcadores de fibroblastos e macrófagos (p=0,007 e p=0,012, respectivamente). As reações da cadeia de polimerase em tempo real da metalanoproteinase-9 e da eritropoietina estavam aumentadas em 2,5 e 6 vezes, respectivamente, em mutantes idosas. A creatinina sérica foi significantemente maior em animais idosos mutantes (p<0,001). Conclusão Essa mutação alterou a arquitetura renal pelo aumento da deposição de matriz extracelular, estresse oxidativo e inflamação, sugerindo papel de proteção de Immp2L contra a fibrose renal. .

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

Novel Combination Markers for Predicting Survival in Patients with Muscle Invasive Bladder Cancer: USP18 and DGCR2

We performed gene expression profiling in bladder cancer patients to identify cancer-specific survival-related genes in muscle invasive bladder cancer (MIBC) patients. Sixty-two patients with MIBC were selected as the original cohort and another 118 MIBC patients were chosen as a validation cohort. The expression of USP18, DGCR2, and ZNF699 genes were measured and we analyzed the association between gene signatures and survival. USP18 and DGCR2, were significantly correlated to cancer-specific death (P=0.020, P=0.007, respectively). Cancer-specific survival in the low USP18 or DGCR2 expression group was significantly longer than the high expression group (P=0.018, P=0.006, respectively). In multivariate Cox regression analysis, a combination of USP18 and DGCR2 mRNA expression levels were significant risk factors for cancer-specific death (HR, 2.106; CI, 1.043-4.254, P=0.038). Overall survival and cancer-specific survival rates in the low-combination group were significantly longer than those in the high-expression group (P=0.001, both). In conclusion, decreased expressions of USP18 and DGCR2 were significantly associated with longer cancer-specific survival, and also the combination of two genes was correlated to a longer survival for MIBC patients. Thus, the combination of USP18 and DGCR2 expression was shown to be a reliable prognostic marker for cancer-specific survival in MIBC.

Expression of the plant viral protease NIa in the brain of a mouse model of Alzheimer's disease mitigates Abeta pathology and improves cognitive function

The plant viral protease, NIa, has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln, with a scissoring property after Gln. We recently reported that NIa efficiently cleaved the amyloid-beta (Abeta) peptide, which contains the sequence Val-His-His-Gln in the vicinity of the cleavage site by alpha-secretase, and that the expression of NIa using a lentiviral system in the brain of AD mouse model reduced plaque deposition levels. In the present study, we investigated whether exogenous expression of NIa in the brain of AD mouse model is beneficial to the improvement of cognitive deficits. To address this question, Lenti-NIa was intracerebrally injected into the brain of Tg-APPswe/PS1dE9 (Tg-APP/PS1) mice at 7 months of age and behavioral tests were performed 15-30 days afterwards. The results of the water maze test indicated that Tg-APP/PS1 mice which had been injected with Lenti-GFP showed an increased latency in finding the hidden-platform and markedly enhanced navigation near the maze-wall, and that such behavioral deficits were significantly reversed in Tg-APP/PS1 mice injected with Lenti-NIa. In the passive avoidance test, Tg-APP/PS1 mice exhibited a severe deficit in their contextual memory retention, which was reversed by NIa expression. In the marble burying test, Tg-APP/PS1 mice buried marbles fewer than non-transgenic mice, which was also significantly improved by NIa. After behavioral tests, it was verified that the Tg-APP/PS1 mice with Lenti-NIa injection had reduced Abeta levels and plaque deposition when compared to Tg-APP/PS1 mice. These results showed that the plant viral protease, NIa, not only reduces Abeta pathology, but also improves behavioral deficits.

Properties of alkaline protease genetically engineered on cell surface of the yeast Yarrowia lipolytica.

ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6 His tag was found to be significantly higher than that of without 6 His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.

Maternal and neonatal screening for Group B streptococci by SCP B gene based PCR: a preliminary study.

OBJECTIVE: To detect the magnitude of group B streptococcal (GBS) colonization and disease among a sample of pregnant women and their infants in Egypt. STUDY DESIGN: Prospective observational study. PARTICIPANTS: The study included 95 pregnant females, 35-37 weeks of gestational age, attending the antenatal outpatient clinic at AlFayom University Hospital between September 2006 and June 2007. All participants were screened with vaginorectal swabs by a conventional GBS PCR assay. Participants were grouped into group A (GBS present, 17 patients) and group B (GBS absent, 78 patients). Details with regard to labor and delivery were recorded and placental pathology was examined to detect histological chorioamnionitis. Ninety-five infant data were also recorded. All neonates of group A (17 out of 95 with known positive maternal GBS) underwent collection of simultaneous specimens from surface sites for PCR before their first bath and within four hours of birth. RESULTS: GBS carriage rate in the study sample was 17.89%. Chorioamnionitis confirmed in three patients by placental pathology (one was in group A and two in group B) was statistically not significant. Twenty-two women had rupture of membranes (< 12 hours) before delivery (four from group A and 18 from group B) that was not statistically significant. There were three infants out of 17 in group A who had GBS colonized at one or more sites by PCR which was statistically significant. However, only one infant was admitted to neonatal intensive care unit (NICU) that was not statistically significant. CONCLUSION: Maternal GBS carriage is associated with a significant increase in neonatal infection rate but is not associated with an increase in neonatal intensive care admission. An accurate evaluation of colonization rate (using a larger sample) is desired to evaluate neonatal invasive disease and determine the cost effectiveness of PCR to select an appropriate preventive strategy in Egypt.

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette.

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies.The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

Clinical diagnosis of group B streptococci by scpB gene based PCR.

BACKGROUND & OBJECTIVES: The goal of the present study was to improve and simplify the diagnosis of Streptococcus agalactiae (group B Streptococcus, GBS) infection for routine clinical practice. METHODS: A total of 71 clinical samples were tested by microbiologic culture, counter immunoelectrophoresis (CIE) and PCR described in the literature. Southern hybridization was accomplished with the Enzo(TM) "DNA Labeling and Detection Kit", Roche (Germany). The computer techniques were used for selection of the specific primers and for analysis of the sizes of PCR products. RESULTS: The primers for the regions around the 51 bp deletion in C5a peptidase gene (scpB) of GBS were selected. PCR analysis revealed the 255 bp amplification fragment in GBS, 306 bp fragment in groups A and G streptococci (GAS, GGS) and did not reveal any fragments in other bacterial species. Among 71 urine and serum clinical samples tested, none were found to be GBS positive by microbiologic culture, 16 samples by CIE, 36 by PCR. The specificity of amplification was confirmed by Southern hybridization. INTERPRETATION & CONCLUSION: The 51 bp deletion in scpB gene in comparison with scpA and scpG genes can be used as a diagnostic tool for identification of GBS. The 51 bp deletion based PCR proved to be faster and more reliable test than microbiologic culture or CIE.

Prevalence of HIV-1 drug resistance in antiretroviral-naive pregnant Thai women.

HIV-1 drug resistance may limit the use of antiretrovirals when attempting to reduce the vertical transmission rate. Establishing the prevalence of the HIV-1 mutations associated with antiretroviral resistance in pregnant women will enable clinicians to maximize the chances of preventing vertical transmission. In order to determine the prevalence of HIV-1 resistant strains among antiretroviral-naive pregnant Thai women, the nucleotide sequences of the HIV-1 polymerase (pol) gene were evaluated. The plasma samples were collected from the women during the 34th week of pregnancy: numerous secondary mutations could be found in the reverse transcriptase (RT) and protease gene, while no primary mutations in the pol gene were found. The result also showed that by detecting the delta32bp deletion within the CCR 5 locus, it was evident that none of HIV-1 infected individuals had homozygous or heterozygous delta32bp deletions of the CCR5 gene; moreover, no CCR5 gene mutations were found in any individual.

Prevalence of primary and secondary resistant mutations to antiretroviral drug in a population of Puerto Rican infected with HIV

INTRODUCTION: Several studies have reported increasing number of therapeutic failures with HAART in HIV-infected individuals. In order to assess the impact HIV antiretroviral resistance could have on treatment, we decided to determine the prevalence of primary and secondary antiretroviral resistant genotypes in a population of HIV-infected Puerto Ricans and compare the mutational distribution pattern with that reported in Europe and US. METHOD: In a total of 80 plasma samples from patients with detectable viral load of over 1,000 RNA copies/ml, the Trugene Visible Genetics HIV sequencing method was used to detect antiretroviral resistance mutations. RESULTS: We found 55 subjects (69 per cent) with high level of resistance to ZDV in the reverse transcriptase gene and 46 subjects (58 per cent) with high level of resistance to NFV in the protease gene. Mutation frequencies to the NRTI ranged in appearance from as high as 54 per cent (i.e., M184V) in the studied subjects to a low of less than 5 per cent (i.e., M184I and V75T). For the NNRTI the most common mutation was K103N in 40 per cent of the subjects and found to confer cross resistance to NVP, DLV and EFV. Another concerning finding is the increasing trend of the frequency of primary and secondary resistant mutations from year 2000 to 20001. Nine (23 per cent) of the total detected primary mutations, to either RTI or PI, showed an increase of at least 5 per cent from one year to the other. Similarly, there were 6 (11 per cent) secondary resistant mutations showing an increase of at least 5 per cent during the two years studied. CONCLUSIONS: In two year period we detected a tendency to increase in primary and secondary HIV-resistant mutation in a population of HIV-infected Puerto Ricans.