The clinical and molecular spectrum of galactosemia in patients from the Cape Town region of South Africa

BACKGROUND: The objective of this study was to document the clinical, laboratory and genetic features of galactosemia in patients from the Cape Town metropolitan region. METHODS: Diagnoses were based on thin layer chromatography for galactosuria/galactosemia and assays of erythrocyte galactose-1-phosphate uridyltransferase (GALT) and galactokinase activities. Patients were screened for the common S135L and Q188R transferase gene mutations, using PCR-based assays. Screening for the S135L mutation in black newborns was used to estimate the carrier rate for galactosemia in black South Africans. RESULTS: A positive diagnosis of galactosemia was made in 17 patients between the years 1980 to 2001. All had very low or absent galactose-1-phosphate uridyltransferase (GALT) activity, and normal galactokinase levels. The mean age at diagnosis was 5.1 months (range 4 days to 6.5 months). A review of 9 patients showed that hepatomegaly (9/9), and splenomegaly, failure to thrive, developmental delay, bilateral cataracts (6/9) were the most frequent features at diagnosis. Six had conjugated hyperbilirubinemia. Four experienced invasive E. coli infection before diagnosis. Ten patients were submitted to DNA analysis. All 4 black patients and 2 of mixed extraction were homozygous for the S135L allele, while all 3 white patients were homozygous for the Q188R allele. The remaining patient of mixed extraction was heterozygous for the Q188R allele. The estimated carrier frequency of the S135L mutation in 725 healthy black newborns was 1/60. CONCLUSIONS: In the absence of newborn screening the delay in diagnosis is most often unacceptably long. Also, carrier frequency data predict a galactosemia incidence of approximately 1/14 400 for black newborns in the Cape Metropole, which is much higher than the current detection rate. It is thus likely that many patients go undetected

Regulatory role of CD8(+ )T lymphocytes in bone marrow eosinophilopoiesis

BACKGROUND: There is a growing body of evidence to suggest that CD8(+ )T lymphocytes contribute to local allergen-induced eosinophilic inflammation. Since bone marrow (BM) responses are intricately involved in the induction of airway eosinophilia, we hypothesized that CD8(+ )T lymphocytes, as well as CD4(+ )T lymphocytes, may be involved in this process. METHODS: Several approaches were utilized. Firstly, mice overexpressing interleukin-5 (IL-5) in CD3(+ )T lymphocytes (NJ.1638; CD3(IL-5+ )mice) were bred with gene knockout mice lacking either CD4(+ )T lymphocytes (CD4(-/-)) or CD8(+ )T lymphocytes (CD8(-/-)) to produce CD3(IL-5+ )knockout mice deficient in CD4(+ )T lymphocytes (CD3(IL-5+)/CD4(-/-)) and CD8(+ )T lymphocytes (CD3(IL-5+)/CD8(-/-)), respectively. Secondly, CD3(+), CD4(+ )and CD8(+ )T lymphocytes from naïve CD3(IL-5+ )and C57BL/6 mice were adoptively transferred to immunodeficient SCID-bg mice to determine their effect on BM eosinophilia. Thirdly, CD3(IL-5+), CD3(IL-5+)/CD8(-/- )and CD3(IL-5+)/CD4(-/- )mice were sensitized and allergen challenged. Bone marrow and blood samples were collected in all experiments. RESULTS: The number of BM eosinophils was significantly reduced in CD3(IL-5+)/CD8(-/- )mice compared to CD3(IL-5+ )mice and CD3(IL-5+)/CD4(-/- )mice. Serum IL-5 was significantly higher in CD3(IL-5+)/CD4(-/- )mice compared to CD3(IL-5+ )mice but there was no difference in serum IL-5 between CD3(IL-5+)/CD4(-/- )and CD3(IL-5+)/CD8(-/- )mice. Adoptive transfer of CD8(+), but not CD4(+ )T lymphocytes from naïve CD3(IL-5+ )and C57BL/6 mice restored BM eosinophilia in immunodeficient SCID-bg mice. Additionally, allergen challenged CD3(IL-5+)/CD8(-/- )mice developed lower numbers of BM eosinophils compared to CD3(IL-5+ )mice and CD3(IL-5+)/CD4(-/- )mice. CONCLUSION: This study shows that CD8(+ )T lymphocytes are intricately involved in the regulation of BM eosinophilopoiesis, both in non-sensitized as well as sensitized and allergen challenged mice

A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype

<p>Abstract</p> <p>Background</p> <p>Many genetic variations of <it>GALK1 </it>have been identified in the patients with galactokinase (GALK1) deficiency. However, the molecular characteristics of <it>GALK1 </it>in individuals with elevated GALK1 activity are relatively unknown.</p> <p>Methods</p> <p>We investigated the relationship between elevated GALK1 activity and the molecular <it>GALK1 </it>gene variations, and the molecular mechanism underlying elevated GALK1 activity. PCR products from 63 subjects, without any attenuation of galactose degradation enzymes, were sequenced to screen for nucleotide alterations in the <it>GALK1 </it>promoter.</p> <p>Results</p> <p>Three nucleotide substitutions were identified: c.-179A>G, c.-27A>C, and c.-22T>C. With respect to the c.-22T>C mutation, GALK1 activity in 13 subjects with the T/C or C/C genotype was significantly higher than those in 50 subjects with the T/T genotype (p < 0.001). The dual luciferase reporter assay in Hep3B cells showed that the luciferase activity with the <it>GALK1 </it>promoter with the c.-22C mutant allele increased approximately 2.5-fold, compared to that with the c.-22T. A specific DNA-protein complex was observed in an electrophoretic mobility shift assay, with slightly higher affinity to c.-22C than to c.-22T.</p> <p>Conclusion</p> <p>The c.-22T>C mutation, which was observed frequently in individuals with elevated GALK1 activity, increased the expression of a reporter gene through enhanced binding of a currently unidentified nuclear protein. These results suggest that the elevated GALK1 activity resulted from enhanced gene expression, due to nucleotide variation within <it>GALK1 </it>promoter.</p

Mirizzi syndrome type IV associated with cholecystocolic fistula: a very rare condition- report of a case

<p>Abstract</p> <p>Background</p> <p>Mirizzi syndrome is a rare complication of prolonged cholelithiasis with presence of large, impacted gallstone into the Hartman's pouch, causing chronic extrinsic compression of common bile duct (CBD). Fistula formation between the CBD and the gallbladder may represent an outcome of that condition. According to Mirizzi's classification and Csendes's subclassification, Mirizzi syndrome type IV represents the most uncommon type (4%).</p> <p>Spontaneous biliary-enteric fistulas have also been rarely reported (1.2–5%) in a large series of cholecystectomies. Cholecystocolic fistula is the most infrequent biliary enteric fistula, causing significant morbidity and representing a diagnostic challenge.</p> <p>Case presentation</p> <p>We describe a very rare, to our knowledge, combination of Mirizzi syndrome type IV and cholecystocolic fistula. A 52 year old male, presented to our clinic complaining of episodic diarrhea (monthly episodes lasting 16 days), high temperature (38°C–39°C), right upper quadrant pain without jaundice. The definitive diagnosis was made intraoperatively. Magnetic Resonance Imaging (MRI) and Endoscopic Retrograde Cholangiopancreatography (ERCP) demonstrated the presence of Mirizzi syndrome with cholecystocolic fistula formation. The patient was operated upon, and cholecystectomy, cholecystocolic fistula excision and Roux-en-Y biliary-enteric anastomosis were undertaken with excellent post-operative course.</p> <p>Conclusion</p> <p>Appropriate biliary tree imaging with ERCP and MRI/MRCP is essential for the diagnosis of Mirizzi syndrome and its complications. Cholecystectomy, fistula excision and biliary-enteric anastomosis with Roux-en-Y loop appears to be the most appropriate surgical intervention in order to avoid damage to Calot's triangle anatomic elements. Particularly in our case, ERCP was a valuable diagnostic tool that Mirizzi syndrome type IV and cholecystocolic fistula.</p

Therapeutic Effects of Autologous Tumor-Derived Nanovesicles on Melanoma Growth and Metastasis

Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Recently, nano-sized vesicles, such as exosomes derived from tumors, were suggested as potential antigen candidates, although the total yield of exosomes is not sufficient for clinical applications. In the present study, we developed a new vaccine strategy based on nano-sized vesicles derived from primary autologous tumors. Through homogenization and sonication of tumor tissues, we achieved high yields of vesicle-bound antigens. These nanovesicles were enriched with antigenic membrane targets but lacked nuclear autoantigens. Furthermore, these nanovesicles together with adjuvant activated dendritic cells in vitro, and induced effective anti-tumor immune responses in both primary and metastatic melanoma mouse models. Therefore, autologous tumor-derived nanovesicles may represent a novel source of antigens with high-level immunogenicity for use in acellular vaccines without compromising safety. Our strategy is cost-effective and can be applied to patient-specific cancer therapeutic vaccination

Preponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases

BACKGROUND: Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions. METHODS: We here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis. RESULTS: Sequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas. The activating mutation T1796A was present in 24/60 (40%) resection specimens, followed in frequency by the oncogenic g1795A mutation in 8/60 (13%) cases. As to the B-raf protein sequence, the acidic amino acid transitions V599E and V599K were predicted in 19/60 (32%) and 6/60 (10%) cases, resepectively, but were not associated with enhanced risk for subsequent metastasis in patients' follow up. In comparison to the primary melanomas that we recently investigated, the spectrum of predicted B-raf protein mutations narrowed significantly in the cutaneous/subcutaneous metastases. Unexpectedly, V599 and V599E mutations were absent in cutaneous/subcutaneous metastases derived from acrolentiginous melanomas as preceding primary tumours. CONCLUSION: During transition from primary melanomas towards cutaneous/subcutaneous metastases, the spectrum of predicted B-raf mutations narrows significantly. Focusing on the V599E and V599K, these oncogenic mutations are likely to affect melanocyte-specific pathways controlling proliferation and differentiation

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO2 fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation

Use of 16S rRNA Gene Based Clone Libraries to Assess Microbial Communities Potentially Involved in Anaerobic Methane Oxidation in a Mediterranean Cold Seep

This study provides data on the diversities of bacterial and archaeal communities in an active methane seep at the Kazan mud volcano in the deep Eastern Mediterranean sea. Layers of varying depths in the Kazan sediments were investigated in terms of (1) chemical parameters and (2) DNA-based microbial population structures. The latter was accomplished by analyzing the sequences of directly amplified 16S rRNA genes, resulting in the phylogenetic analysis of the prokaryotic communities. Sequences of organisms potentially associated with processes such as anaerobic methane oxidation and sulfate reduction were thus identified. Overall, the sediment layers revealed the presence of sequences of quite diverse bacterial and archaeal communities, which varied considerably with depth. Dominant types revealed in these communities are known as key organisms involved in the following processes: (1) anaerobic methane oxidation and sulfate reduction, (2) sulfide oxidation, and (3) a range of (aerobic) heterotrophic processes. In the communities in the lowest sediment layer sampled (22–34 cm), sulfate-reducing bacteria and archaea of the ANME-2 cluster (likely involved in anaerobic methane oxidation) were prevalent, whereas heterotrophic organisms abounded in the top sediment layer (0–6 cm). Communities in the middle layer (6–22 cm) contained organisms that could be linked to either of the aforementioned processes. We discuss how these phylogeny (sequence)-based findings can support the ongoing molecular work aimed at unraveling both the functioning and the functional diversities of the communities under study

Drug Off-Target Effects Predicted Using Structural Analysis in the Context of a Metabolic Network Model

Recent advances in structural bioinformatics have enabled the prediction of protein-drug off-targets based on their ligand binding sites. Concurrent developments in systems biology allow for prediction of the functional effects of system perturbations using large-scale network models. Integration of these two capabilities provides a framework for evaluating metabolic drug response phenotypes in silico. This combined approach was applied to investigate the hypertensive side effect of the cholesteryl ester transfer protein inhibitor torcetrapib in the context of human renal function. A metabolic kidney model was generated in which to simulate drug treatment. Causal drug off-targets were predicted that have previously been observed to impact renal function in gene-deficient patients and may play a role in the adverse side effects observed in clinical trials. Genetic risk factors for drug treatment were also predicted that correspond to both characterized and unknown renal metabolic disorders as well as cryptic genetic deficiencies that are not expected to exhibit a renal disorder phenotype except under drug treatment. This study represents a novel integration of structural and systems biology and a first step towards computational systems medicine. The methodology introduced herein has important implications for drug development and personalized medicine