FANCM Gene Variants in a Male Diagnosed with Sertoli Cell-Only Syndrome and Diffuse Astrocytoma.

Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

Effects of S-Adenosylhomocysteine Hydrolase Downregulation on Wnt Signaling Pathway in SW480 Cells.

S-adenosylhomocysteine hydrolase (AHCY) deficiency results mainly in hypermethioninemia, developmental delay, and is potentially fatal. In order to shed new light on molecular aspects of AHCY deficiency, in particular any changes at transcriptome level, we enabled knockdown of AHCY expression in the colon cancer cell line SW480 to simulate the environment occurring in AHCY deficient individuals. The SW480 cell line is well known for elevated AHCY expression, and thereby represents a suitable model system, in particular as AHCY expression is regulated by MYC, which, on the other hand, is involved in Wnt signaling and the regulation of Wnt-related genes, such as the ß-catenin co-transcription factor LEF1 (lymphoid enhancer-binding factor 1). We selected LEF1 as a potential target to investigate its association with S-adenosylhomocysteine hydrolase deficiency. This decision was prompted by our analysis of RNA-Seq data, which revealed significant changes in the expression of genes related to the Wnt signaling pathway and genes involved in processes responsible for epithelial-mesenchymal transition (EMT) and cell proliferation. Notably, LEF1 emerged as a common factor in these processes, showing increased expression both on mRNA and protein levels. Additionally, we show alterations in interconnected signaling pathways linked to LEF1, causing gene expression changes with broad effects on cell cycle regulation, tumor microenvironment, and implications to cell invasion and metastasis. In summary, we provide a new link between AHCY deficiency and LEF1 serving as a mediator of changes to the Wnt signaling pathway, thereby indicating potential connections of AHCY expression and cancer cell phenotype, as Wnt signaling is frequently associated with cancer development, including colorectal cancer (CRC).

HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia.

IMPORTANCE: Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.

Association of NLRP1 Coding Polymorphism with Lung Function and Serum IL-1ß Concentration in Patients Diagnosed with Chronic Obstructive Pulmonary Disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a chronic disease characterized by a progressive decline in lung function due to airflow limitation, mainly related to IL-1ß-induced inflammation. We have hypothesized that single nucleotide polymorphisms (SNPs) in NLRP genes, coding for key regulators of IL-1ß, are associated with pathogenesis and clinical phenotypes of COPD. We recruited 704 COPD individuals and 1238 healthy controls for this study. Twenty non-synonymous SNPs in 10 different NLRP genes were genotyped. Genetic associations were estimated using logistic regression, adjusting for age, gender, and smoking history. The impact of genotypes on patients' overall survival was analyzed with the Kaplan-Meier method with the log-rank test. Serum IL-1ß concentration was determined by high sensitivity assay and expression analysis was done by RT-PCR. Decreased lung function, measured by a forced expiratory volume in 1 s (FEV1% predicted), was significantly associated with the minor allele genotypes (AT + TT) of NLRP1 rs12150220 (p = 0.0002). The same rs12150220 genotypes exhibited a higher level of serum IL-1ß compared to the AA genotype (p = 0.027) in COPD patients. NLRP8 rs306481 minor allele genotypes (AG + AA) were more common in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) definition of group A (p = 0.0083). Polymorphisms in NLRP1 (rs12150220; OR = 0.55, p = 0.03) and NLRP4 (rs12462372; OR = 0.36, p = 0.03) were only nominally associated with COPD risk. In conclusion, coding polymorphisms in NLRP1 rs12150220 show an association with COPD disease severity, indicating that the fine-tuning of the NLRP1 inflammasome could be important in maintaining lung tissue integrity and treating the chronic inflammation of airways.

Promoter methylation status of ASC/TMS1/PYCARD is associated with decreased overall survival and TNM status in patients with early stage non-small cell lung cancer (NSCLC).

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, with 5-year overall survival less than 15%. Therefore, it is essential to find biomarkers for early detection and prognosis. Aberrant DNA methylation is a common feature of human cancers and its utility is already recognized in cancer management. The aim of this study was to explore the diagnostic and prognostic value of the promoter methylation status of the ASC/TMS1/PYCARD and MyD88 genes, key adaptor molecules in the activation of the innate immune response and apoptosis pathways. METHODS: A total of 50 non-small cell lung cancer (NSCLC) patients were enrolled in the study. Methylation of bisulphite converted DNA was quantified by pyrosequencing in fresh frozen malignant tissues and adjacent non-malignant tissues. Associations between methylation and lung function, tumor grade and overall survival were evaluated using receiver-operating characteristics (ROC) analysis and statistical tests of hypothesis. RESULTS: Methylation level of tested genes is generally low but significantly decreased in tumor tissues (ASC/TMS1/PYCARD, P<0.0001; MyD88, P<0.0002), which correlates with increased protein expression. Three CpG sites were identified as promising diagnostic marker candidates; CpG11 (-63 position) in ASC/TMS1/PYCARD and CpG1 (-253 position) and 2 (-265 position) in MyD88. The association study showed that the methylation status of the ASC/TMS1 CpG4 site (-34 position) in malignant and non-malignant tissues is associated with the overall survival (P=0.019) and the methylation status of CpG8 site (-92 position) is associated with TNM-stage (P=0.011). CONCLUSIONS: The methylation status of the ASC/TMS1/PYCARD and MyD88 promoters are promising prognostic biomarker candidates. However, presented results should be considered as a preliminary and should be confirmed on the larger number of the samples.

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest.

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

A Proximity Extension Assay (PEA)-based method for quantification of bevacizumab.

INTRODUCTION: Proximity Extension Assay (PEA) is a direct one-step protein quantification method using a pair of DNA oligonucleotides linked to antibodies against the target molecule. It requires polyclonal or two monoclonal antibodies (mAbs) that bind to target epitopes close enough to form a DNA duplex which is quantified by real-time PCR. Bevacizumab, an anti-cancer drug, is a mAb against vascular endothelial growth factor with common cardiovascular adverse effects. It is widely used off-label to treat neovascular eye disorders by intravitreal application of small doses. Even then, certain amount reaches systemic circulation which is considered relevant regarding safety. We aimed to set-up a PEA-based assay for bevacizumab in human plasma and to preliminary evaluate it in patients treated intravitreally. METHODS: We tested (PEA, quantitative PCR) several combinations of commercial mAbs and a Fab fragment against bevacizumab. The best combination was used to quantify bevacizumab in three patients donating plasma before and 24 h after the first intravitreal injection. RESULTS: A combination of a mAb and a Fab fragment (HCA184 and HCA182, Bio-Rad Laboratories, Inc.) performed best: standard curve R2 0.98, linear dynamic range 1-1000 pM, lower limit of quantification 1 pM (149 pg/mL) and a satisfactory precision (coefficient of variation 12%). All pre-dose patient concentrations were zero, while post-dose concentrations were 10.94, 13.73 and 55.49 ng/mL, in line with previous reports. DISCUSSION: This is the first set-up of a PEA-based assay for quantification of bevacizumab in human plasma. Its good performance and high sensitivity support further evaluation for potential uses particularly when the expected concentrations are low.

Mutations in S-adenosylhomocysteine hydrolase (AHCY) affect its nucleocytoplasmic distribution and capability to interact with S-adenosylhomocysteine hydrolase-like 1 protein.

S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.

1-ethyl-3-(6-methylphenanthridine-8-il) urea modulates TLR3/9 activation and induces selective pro-inflammatory cytokine expression in vitro.

We have previously demonstrated the nucleic acid binding capacity of phenanthridine derivatives (PHTs). Because nucleic acids are potent inducers of innate immune response through Toll-like receptors (TLRs), and because PTHs bear a structural resemblance to commonly used synthetic ligands for TLR7/8, we hypothesized that PHTs could modulate/activate immune response. We found that compound M199 induces secretion of IL-6, IL-8 and TNFα in human PBMCs and inhibits TLR3/9 activation in different cellular systems (PBMCs, HEK293 and THP-1 cell lines).

Combining Unique Multiplex Gateway Cloning and Bimolecular Fluorescence Complementation (BiFC) for High-Throughput Screening of Protein-Protein Interactions.

Protein interaction networks are the basis for human metabolic and signaling systems. Interaction studies often use bimolecular fluorescence complementation (BiFC) to reveal the formation and cellular localization of protein complexes. However, large-scale studies were either far from native conditions in human cells or limited by laborious restriction/ligation cloning techniques. Here, we describe a new tool for protein interaction screening based on Gateway-compatible BiFC vectors. We made a set of four new vectors that permit fusion of candidate proteins to the N or C fragment of Venus in all fusion positions. We have validated the vectors and confirmed self-association of AHCY, AHCYL1, and galectin-3. In a high-throughput BiFC screen, we identified new AHCY interaction partners: galectin-3 and PUS7L. We also describe additional steps in protein interaction analysis, applied for AHCY-galectin-3 interaction. First, we classified the interaction in intracellular vesicles using CellCognition, machine learning free software. Then we identified the vesicles as endosomal pathway compartments, in line with known galectin-3 trafficking route. This offers a platform to rapidly identify and localize new protein interactions inside living cells, a prerequisite to validate in silico interactome data, and ultimately decode complex protein networks.

Abnormal Hypermethylation at Imprinting Control Regions in Patients with S-Adenosylhomocysteine Hydrolase (AHCY) Deficiency.

S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status.

Liver transplantation for treatment of severe S-adenosylhomocysteine hydrolase deficiency.

A child with severe S-adenosylhomocysteine hydrolase (AHCY) deficiency (AHCY c.428A>G, p.Tyr143Cys; c.982T>G, p.Tyr328Asp) presented at 8 months of age with growth failure, microcephaly, global developmental delay, myopathy, hepatopathy, and factor VII deficiency. Plasma methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) were markedly elevated and the molar concentration ratio of AdoMet:AdoHcy, believed to regulate a myriad of methyltransferase reactions, was 15% of the control mean. Dietary therapy failed to normalize biochemical markers or alter the AdoMet to AdoHcy molar concentration ratio. At 40 months of age, the proband received a liver segment from a healthy, unrelated living donor. Mean AdoHcy decreased 96% and the AdoMet:AdoHcy concentration ratio improved from 0.52±0.19 to 1.48±0.79 mol:mol (control 4.10±2.11 mol:mol). Blood methionine and AdoMet were normal and stable during 6 months of follow-up on an unrestricted diet. Average calculated tissue methyltransferase activity increased from 43±26% to 60±22%, accompanied by signs of increased transmethylation in vivo. Factor VII activity increased from 12% to 100%. During 6 postoperative months, head growth accelerated 4-fold and the patient made promising gains in gross motor, language, and social skills.

Probing the structural properties of DNA/RNA grooves with sterically restricted phosphonium dyes: screening of dye cytotoxicity and uptake.

To explore in greater detail the recently reported rare kinetic differentiation between homo-polymeric and alternating AT-DNA sequences by using sterically restricted phosphonium dyes that form dimers within the DNA minor groove, new analogues were prepared in which the quinolone phosphonium moiety was kept constant, while the size and hydrogen bonding properties of the rest of the molecule were varied. Structure-activity relationship studies revealed that a slight increase in length by an additional methylene unit results in loss of kinetic AT selectivity, but yielded an AT-selective fluorescence response. These DNA/RNA-groove-bound dyes combine very low cytotoxicity with efficient cellular uptake and intriguingly specific fluorescent marking of mitochondria. In contrast to longer analogues, a decrease in length (by methylene unit removal) and rearrangement of positive charge resulted in dyes that had switched to the intercalative binding mode to GC DNA/dsRNA but that still form dimers in the minor groove of AT sequences, consequently yielding a significantly different chiro-optical response. The latter dyes also revealed strongly selective antiproliferative activity toward HeLa cancer cells.

Clinical picture of S-adenosylhomocysteine hydrolase deficiency resembles phosphomannomutase 2 deficiency.

We report on the seventh known patient with S-adenosylhomocysteine hydrolase (SAHH) deficiency presenting at birth with features resembling phosphomannomutase 2 (PMM2-CDG Ia) deficiency. Plasma methionine and total homocysteine levels were normal at 2 months and increased only after the 8th month of age. SAHH deficiency was confirmed at 4.5 years of age by showing decreased SAHH activity (11% in both erythrocytes and fibroblasts), and compound heterozygosity for a known mutation c.145C>T (p.R49C) and a novel variant c.211G>A (p.G71S) in the AHCY gene. Retrospective analysis of clinical features revealed striking similarities between SAHH deficiency and the PMM2-CDG Ia.

S-adenosylhomocysteine hydrolase deficiency: two siblings with fetal hydrops and fatal outcomes.

This paper reports the clinical and metabolic findings in two sibling sisters born with fetal hydrops and eventually found to have deficient S-adenosylhomocysteine hydrolase (AHCY) activity due to compound heterozygosity for two novel mutations, c.145C>T; p.Arg49Cys and c.257A>G; p.Asp86Gly. Clinically, the major abnormalities in addition to fetal hydrops (very likely due to impaired synthetic liver function) were severe hypotonia/myopathy, feeding problems, and respiratory failure. Metabolic abnormalities included elevated plasma S-adenosylhomocysteine, S-adenosylmethionine, and methionine, with hypoalbuminemia, coagulopathies, and serum transaminase elevation. The older sister died at age 25 days, but the definitive diagnosis was made only retrospectively. The underlying genetic abnormality was diagnosed in the second sister, but treatment by means of dietary methionine restriction and supplementation with phosphatidylcholine and creatine did not prevent her death at age 122 days. These cases extend the experience with AHCY deficiency in humans, based until now on only the four patients previously identified, and suggest that the deficiency in question may be a cause of fetal hydrops and developmental abnormalities of the brain.

S-adenosylhomocysteine hydrolase (AHCY) deficiency: two novel mutations with lethal outcome.

This paper reports studies of two novel, allelic missense mutations found in the S-adenosylhomocysteine hydrolase (AHCY) gene from a new case of AHCY deficiency in an infant girl who died at age four months. The mutations lead to replacement of arginine with cysteine (p.Arg49Cys) and aspartic acid with glycine (p.Asp86Gly). Functional analysis of recombinant proteins containing the mutations detected showed that both dramatically reduce AHCY activity. The p.Arg49Cys mutant protein forms intermolecular disulphide bonds, leading to macromolecular structures that can be prevented by reducing agent DTT. The p.Asp86Gly protein tends to form enzymatically inactive aggregates and the loss of a single negative charge as a result of the mutation is involved in enzyme inactivation. We show that replacing Gly86 with negatively charged Glu86 in mutant protein restores enzymatic activity to 70% of wild-type, whereas changing Gly86 to positively charged Lys86 or uncharged Leu86 does not improve enzyme activity, indicating that the negative charge is important for maintenance of such activity. These studies significantly extend knowledge about the importance of residue 86 for AHCY activity. Residue 86 has not been implicated before in this way and the results suggest that the present model of S- adenosylhomocysteine (AdoHcy) hydrolysis may need refinement. Our functional studies provide novel insight into the molecular defect underlying AHCY deficiency and reveal that both low enzyme activity and protein stability of AHCY contribute to the clinical phenotype.

Functional analysis of human S-adenosylhomocysteine hydrolase isoforms SAHH-2 and SAHH-3.

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of AdoHcy to adenosine and homocysteine. Increased levels of AdoHcy may play a role in the development of cardiovascular diseases and numerous other conditions associated with hyperhomocysteinemia. Several polymorphic isoforms named SAHH-1 to 4 may be resolved by horizontal starch gel electrophoresis from red blood cells. We have identified the genetic background of isoforms SAHH-2 and SAHH-3. SAHH-2 represents the previously described polymorphism in exon 2 of the AdoHcyase gene (112 C>T; p.R38W). Isoform SAHH-3 is based on a new polymorphism in exon 3 (377 G>A), leading to the conversion of glycine to arginine at amino-acid position 123. To shed light on the effects of these polymorphisms on the molecular and catalytic properties of AdoHcyase, we made recombinant wild-type and polymorphic R38W and G123R enzymes for a comparative analysis. The amino-acid exchanges did not bring about major changes to the catalytic rates of the recombinant proteins. However, circular dichroism analysis showed that both polymorphisms effect the thermal stability of the recombinant protein in vitro, reducing the unfolding temperature by approximately 2.6 degrees C (R38W) and 1.5 degrees C (G123R) compared to wild-type protein. In view of the altered thermal stability, and slightly decreased enzymatic activity of polymorphic proteins (< or =6%), one may consider the analyzed AdoHcyase isoforms as risk markers for diseases caused by irregular AdoHcyase metabolism.

A single mutation at Tyr143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and affects the oxidation state of bound cofactor nicotinamide-adenine dinucleotide.

Recently, we have described the first human case of AdoHcyase (S-adenosylhomocysteine hydrolase) deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) and the truncation mutation p.W112stop (AdoHcyase in which Trp112 is replaced by opal stop codon) were identified [Baric, Fumic, Glenn, Cuk, Schulze, Finkelstein, James, Mejaski-Bosnjak, Pazanin, Pogribny et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 4234-4239]. To elucidate the molecular and catalytic properties of AdoHcyase, we have made recombinant wild-type and mutant p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased from 65% to 75%. Further, the oxidation states of coenzyme NAD differ between mutant and wild-type protein, with an increased NADH accumulation in the mutant p.Y143C enzyme of 88% NADH (wild-type contains 18% NADH). Quantitative binding of NAD is not affected. Native polyacrylamide gel electrophoresis showed, that mutant p.Y143C subunits are able to form the tetrameric complex as is the wild-type enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7 degrees C compared with wild-type protein. Change of Glu115 to lysine in wild-type protein causes a change in thermosensitivity almost identical with that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Tyr143 and Glu115. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the AdoHcyase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with AdoHcyase deficiency.

S-adenosylhomocysteine hydrolase deficiency in a human: a genetic disorder of methionine metabolism.

We report studies of a Croatian boy, a proven case of human S-adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 microM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5-15.9 microM. In plasma, S-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was approximately equal to 3% of control in liver and was 5-10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.

Class XIII myosins from the green alga Acetabularia: driving force in organelle transport and tip growth?

The green alga Acetabularia cliftonii (Dasycladales) contains at least two myosin genes, which already have been assigned class XIII of the myosin superfamily (Cope et al., 1996, Structure 4: 969-987). Here we report a complete analysis of their gene structure and their corresponding transcripts Aclmyo1 and Aclmyo2. Despite promising Northern blot data no evidence for alternative splicing could be found. Dissecting the primary structure at complementary deoxyribonucleic acid (cDNA) level we found a myosin typical organization in head, neck and variable tail region. Most striking is the extremely short tail region of Aclmyo1 with only 18 residues and the maximum number of 7 IQ motifs in Aclmyo2. Probing Acetabularia protein extracts with an antibody raised to a synthetic peptide derived from the amino terminal region in Alcmyo1 showed cross-reactivity to a polypeptide with a molecular mass of approximately 100 kD. This corresponds to the predicted molecular weight of Aclmyo1, which is 106 kD as deduced from the amino acid sequence. Additionally, the same cross-reactive protein is capable of binding F-actin as indicated by a co-sedimentation assay. Confocal laser scanning microscopy with raised antibody revealed co-localization with organelles, the budding region of lateral whorls and the cell apex suggesting involvement of putative Acetabularia myosin in organelle transport and tip growth.