Neurochemical Characterization of Canine α-L-Iduronidase Deficiency Disease (Model of Human Mucopolysaccharidosis I)

This report presents the neurochemical findings on the first dog to die with deficiency of alpha-L-iduronidase (mucopolysaccharide alpha-L-iduronohydrolase; EC 3.2.1.76). The principal findings were (a) markedly increased glycosaminoglycan content in all neural tissues examined (from threefold in sciatic nerve to 15-fold in brainstem), (b) a modest increase in levels of gangliosides GM2, GM3, and GD3, particularly in gray matter, (c) excessive accumulation of glycosaminoglycans in the CSF, (d) the increased glycosaminoglycans were dermatan sulfate and heparan sulfate, and (e) the molecular weights of the liver glycosaminoglycans were shifted toward smaller sizes, indicating partial degradation. The canine disorder thus resembles mucopolysaccharidosis I in all aspects.     

Effects of Enzyme Replacement Therapy Started Late in a Murine Model of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don’t receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6–8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible.     

Pentosan Polysulfate: Oral Versus Subcutaneous Injection in Mucopolysaccharidosis Type I Dogs

BackgroundWe previously demonstrated the therapeutic benefits of pentosan polysulfate (PPS) in a rat model of mucopolysaccharidosis (MPS) type VI. Reduction of inflammation, reduction of glycosaminoglycan (GAG) storage, and improvement in the skeletal phenotype were shown. Herein, we evaluate the long-term safety and therapeutic effects of PPS in a large animal model of a different MPS type, MPS I dogs. We focused on the arterial phenotype since this is one of the most consistent and clinically significant features of the model.ConclusionsPPS treatment led to reductions of pro-inflammatory cytokines and GAG storage in urine and tissues of MPS I dogs, which were most evident after subQ administration. SubQ administration also led to significant cytokine reductions in the CSF. Both treatment groups exhibited markedly reduced carotid and aortic inflammation, increased vessel integrity, and improved histopathology. We conclude that PPS may be a safe and useful therapy for MPS I, either as an adjunct or as a stand-alone treatment that reduces inflammation and GAG storage.     

Immune-Mediated Inflammation May Contribute to the Pathogenesis of Cardiovascular Disease in Mucopolysaccharidosis Type I

Background

Cardiovascular disease, a progressive manifestation of α-L-iduronidase deficiency or mucopolysaccharidosis type I, continues in patients both untreated and treated with hematopoietic stem cell transplantation or intravenous enzyme replacement. Few studies have examined the effects of α-L-iduronidase deficiency and subsequent glycosaminoglycan storage upon arterial gene expression to understand the pathogenesis of cardiovascular disease.

Methods

Gene expression in carotid artery, ascending, and descending aortas from four non-tolerized, non-enzyme treated 19 month-old mucopolysaccharidosis type I dogs was compared with expression in corresponding vascular segments from three normal, age-matched dogs. Data were analyzed using R and whole genome network correlation analysis, a bias-free method of categorizing expression level and significance into discrete modules. Genes were further categorized based on module-trait relationships. Expression of clusterin, a protein implicated in other etiologies of cardiovascular disease, was assessed in canine and murine mucopolysaccharidosis type I aortas via Western blot and in situ immunohistochemistry.

Results

Gene families with more than two-fold, significant increased expression involved lysosomal function, proteasome function, and immune regulation. Significantly downregulated genes were related to cellular adhesion, cytoskeletal elements, and calcium regulation. Clusterin gene overexpression (9-fold) and protein overexpression (1.3 to 1.62-fold) was confirmed and located specifically in arterial plaques of mucopolysaccharidosis-affected dogs and mice.

Conclusions

Overexpression of lysosomal and proteasomal-related genes are expected responses to cellular stress induced by lysosomal storage in mucopolysaccharidosis type I. Upregulation of immunity-related genes implicates the potential involvement of glycosaminoglycan-induced inflammation in the pathogenesis of mucopolysaccharidosis-related arterial disease, for which clusterin represents a potential biomarker.     

Glycosaminoglycan Storage in Cultured Neonatal Murine Mucopolysaccharidosis Type VII Neuroglial Cells and Correction by β-Glucuronidase Gene Transfer

Abstract: The inherited deficiency of β-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. β-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal β-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of β-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.     

Type I Interferon Controls Propagation of Long Interspersed Element-1

Type I interferons (IFN) including IFNα and IFNβ are critical for the cellular defense against viruses. Here we report that increased levels of IFNβ were found in testes from mice deficient in MOV10L1, a germ cell-specific RNA helicase that plays a key role in limiting the propagation of retrotransposons including Long Interspersed Element-1 (LINE-1). Additional experiments revealed that activation of LINE-1 retrotransposons increases the expression of IFNβ and of IFN-stimulated genes. Conversely, pretreatment of cells with IFN suppressed the replication of LINE-1. Furthermore, the efficacy of LINE-1 replication was increased in isogenic cell lines harboring inactivating mutations in diverse elements of the IFN signaling pathway. Knockdown of the IFN receptor chain IFNAR1 also stimulated LINE-1 propagation in vitro. Finally, a greater accumulation of LINE-1 was found in mice that lack IFNAR1 compared with wild type mice. We propose that LINE-1-induced IFN plays an important role in restricting LINE-1 propagation and discuss the putative role of IFN in preserving the genome stability.     

Recovery of a Canine Herpesvirus from Primary Kidney Cultures Derived from a Closed Dog Colony

The recovery of a canine herpesvirus from one of eight lots of primary canine kidney cultures derived from a closed dog colony is reported.     

Differential Regulation of Type I Interferon and Epidermal Growth Factor Pathways by a Human Respirovirus Virulence Factor

A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.     

Extreme Beta-Cell Deficiency in Pancreata of Dogs with Canine Diabetes

The pathophysiology of canine diabetes remains poorly understood, in part due to enigmatic clinical features and the lack of detailed histopathology studies. Canine diabetes, similar to human type 1 diabetes, is frequently associated with diabetic ketoacidosis at onset or after insulin omission. However, notable differences exist. Whereas human type 1 diabetes often occurs in children, canine diabetes is typically described in middle age to elderly dogs. Many competing theories have been proposed regarding the underlying cause of canine diabetes, from pancreatic atrophy to chronic pancreatitis to autoimmune mediated β-cell destruction. It remains unclear to what extent β-cell loss contributes to canine diabetes, as precise quantifications of islet morphometry have not been performed. We used high-throughput microscopy and automated image processing to characterize islet histology in a large collection of pancreata of diabetic dogs. Diabetic pancreata displayed a profound reduction in β-cells and islet endocrine cells. Unlike humans, canine non-diabetic islets are largely comprised of β-cells. Very few β-cells remained in islets of diabetic dogs, even in pancreata from new onset cases. Similarly, total islet endocrine cell number was sharply reduced in diabetic dogs. No compensatory proliferation or lymphocyte infiltration was detected. The majority of pancreata had no evidence of pancreatitis. Thus, canine diabetes is associated with extreme β-cell deficiency in both new and longstanding disease. The β-cell predominant composition of canine islets and the near-total absence of β-cells in new onset elderly diabetic dogs strongly implies that similar to human type 1 diabetes, β-cell loss underlies the pathophysiology of canine diabetes.     

Novel Pathologic Findings Associated with Urinary Retention in a Mouse Model of Mucopolysaccharidosis Type IIIB

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome type B) is a metabolic disorder with devastating clinical characteristics starting in early childhood and leading to premature death. A knockout mouse strain was developed that models this disease. Mice of the strain B6.129S6- Naglu^tm1Efn/J are invaluable for investigating pathogenesis and possible treatment modalities. However, the mouse strain also exhibits some objectionable phenotypic features. One such feature, urinary retention, not only is atypical of human MPS IIIB but often leads to early termination of experiments due to animal welfare concerns. The aim of this study was to investigate abnormalities associated with the urinary retention. Necropsies were performed on 9-mo-old mice; urinalysis, hematology and blood chemistry parameters were evaluated, and urogenital specimens were microscopically examined. Histopathologic examinations of urinary tract specimens proved illuminating regarding pathology in the urinary tract. A large mononuclear cell infiltrate was discovered in mutant mice of both sexes, more pronounced in females compared with male mice. The infiltrate comprises of large rounded or polygonal cells with generous variably vacuolated, granular eosinophilic cytoplasm and small round vesicular nuclei. These cells were present throughout and expand the interstitium of the lower urinary tract. Either this results in extrinsic compression of the lumen of the urethra, eventually leading to obstructive uropathy, bladder hyperdistension, and urinary retention or possibly interferes with the neurogenic component of micturition needs to be further investigated. The novel finding of an unexpected mononuclear cell infiltrate in the urinary tract in the knockout mice B6.129S6- Naglu^tm1Efn/J is reported.Abbreviations: BSA, bovine serum albumin; BUN, blood urea nitrogen; MPS III B, mucopolysaccharidosis type III BMucopolysaccharidosis type IIIB, also called Sanfilippo syndrome type B, is a metabolic disorder with devastating clinical characteristics in humans. The onset of the disease is usually between 2 and 4 y of age; clinical symptoms, including hyperactivity, aggressive behavior, hearing and vision defects, mental retardation, and mild somatic changes progress rapidly and are followed by premature death, generally in the second decade of life.^3,24 The pathogenesis of MPS IIIB can be described as a lysosomal storage disorder resulting from failure to degrade the lysosomal glycosaminoglycan heparan sulfate due to absence of the enzyme α-N-acetylglucosaminidase (Naglu). This inherited disorder is elicited by mutations in the Naglu gene, which is located on chromosome 17q21.³ More than 85 mutations have been identified so far.The B6.129S6- Naglu^tm1Efn/J mouse strain was developed through targeted mutation by disruption of exon 6 of the Naglu gene.²⁰ These mice are invaluable to continued investigations of pathogenesis of MPS IIIB, possible clinical interventions, and an eventual cure for this devastating disease. However, in addition to a number of desirable characteristics, B6.129S6- Naglu^tm1Efn/J mice exhibit several objectionable phenotypic features.¹² One such adverse feature is urinary retention, leading to a grossly enlarged urinary bladder, potential hydronephrosis, and uremia. This specific abnormality of the genetically engineered mice not only is atypical of human MPS IIIB but due to its effect on animal welfare may lead to early termination of experiments.Urinary retention leading to overdistension of the urinary bladder is a consequence of altered micturition due to urinary incontinence, dysuria, or a combination of both. Micturition is normally a conscious, voluntary act, whereas urinary incontinence is distinguished by the loss of the voluntary management of urination. Dysuria, however, relates to difficult and painful voiding of urine.¹⁹ Both disorders of micturition can cause moderate to severe distension of the bladder either as an acute or chronic condition. Dysuria may be elicited by any obstructive uropathy, and urinary incontinence can be classified as neurogenic or nonneurogenic.¹Bladder distension in multiple strains of mice has been described as a sequela of the toxicity of various substances,^17,25,32 in studies of urinary tract malformations,²⁶ after infection,⁶ and a characteristic of various transgenic mouse strains with various pathogenic pathways, such as mice with a mutated preprotachykinin gene, hypersensitive serotonin 3A receptor mutant mice, and mice lacking the muscarinic receptors M2 and M3.^4,16,23 In addition, urinary retention with bladder enlargement comprises a part of the mouse urologic syndrome, which develops spontaneously in aged mice^15,28 and has only been reported to occur in male mice. The pathogenesis of mouse urologic syndrome is unclear.In our study colony of B6.129S6- Naglu^tm1Efn/J mice, most exhibited moderate to severe enlargement of the urinary bladder in a sex-independent fashion, whereas control (wildtype) mice had a normal or, in a few cases only, slightly enlarged bladder.¹² As mentioned previously, bladder enlargement was the most common cause of euthanasia for animal-welfare reasons, thereby decreasing the data collection time.The aim of the present study was to investigate the clinical and pathologic correlations underlying anomalous bladder enlargement in a mouse model of MPS IIIB. In addition, we attempted to determine whether the observed urinary retention was dependent on the genetic mutation or background strain used, to facilitate interpretation of results from treatment development studies, interpretation which might otherwise be difficult or impossible.     

Pathology of Ground Squirrels in a Research Colony

This paper presents a retrospective review of the postmortem findings in a colony of wild-caught ground squirrels used in medical research. The species included in this study were Richardson''s ground squirrel Urocitellus richardsonii, Columbian ground squirrel Urocitellus columbianus and golden-mantled ground squirrel Callospermophilus lateralis. The pathologic findings in 160 ground squirrels from this colony demonstrated a wide variety of conditions, with chronic nephritis and hepatic adenomas being the most frequent overall. All animals with gross lesions of chronic interstitial nephritis had both glomerular and tubulointerstitial disease upon microscopic examination. As the first review of pathology in a research colony of ground squirrels. this study provides data for use in comparative studies about rodent diseases and important information for those who maintain such animals for research.

Ground squirrels are commonly but incorrectly referred to as gophers and are terrestrial members of the Order Rodentia, family Sciuridae. True gophers are members of the family Geomyidae. In the Canadian province of Alberta, the 3 most common species of ground squirrels are Richardson ground squirrel (Urocitellus richardsonii),^4,13 the Columbian ground squirrel (Urocitellus columbianus),⁵ and the golden-mantled ground squirrel (Callospermophilus lateralis).⁷ Thirteen lined (Ictidomys tridecemlineatus) and Franklin (Poliocitellus franklinii)⁶ ground squirrels are also present but less numerous in Alberta.The 3 species in this study vary in lifespan, litter size, population density,⁵ annual mortality rate and timing of reproduction,¹³ but have several common attributes. An overview of these ground squirrels in the wild is as follows. In a natural habitat, which includes open sagebrush, grasslands,⁴ and mountain meadows, ground squirrels live in complex burrows on stable,⁴ sandy, well-drained soils^4,5 at elevations from 1828 to over 3658 meters in the western 2-thirds of North America.¹ Litters are born in late April to early May and consist of 6 to 8 pups. Ground squirrels are social animals, with densities ranging from 5 to 60 per hectare. In the wild, mortality among male Richardson ground squirrels is as much as 80% per year, partly due to heavy predation.¹⁴ Females have a slightly lower annual mortality rate and are considered to have reached old age at 3 to 4 y of age.¹⁴ One study in southern Alberta found 5 and 6 y old females were present but only a single male that had survived to 4 y.¹³ Ground squirrel burrows can damage to agricultural land because their holes are a hazard to livestock, their mounds can damage equipment, and burrowing can damage crops.¹¹ For these reasons, they are often considered a pest species. On the other hand, ground squirrels till the soil and can serve as a food source for predators.¹¹ As such, they are an integral part of the prairie and mountain ecosystem and food chain.Ground squirrels are most often used to study hibernation and metabolism, as their basal metabolic rate varies according to environmental conditions. They have large amounts of brown fat and are deep seasonal hibernators.³ Unlike most rodents, they are strictly diurnal with peak activities in the morning and late afternoon. Hibernation in ground squirrels consists of periods of torpor interspersed with brief thermogenic arousals that last less than 24 h. Torpor bouts increase in length with decreasing soil temperature. During periods of inactivity, body temperature drops to near ambient soil temperature, falling as low as 2 to 3 °C in late winter.¹² Despite the metabolic costs of thermogenesis and euthermy during intertorpor arousals, hibernating ground squirrels can achieve an overall energy saving over the hibernating season of 88% compared with euthermic animals.¹⁹ Complex regulation of body temperature makes ground squirrels ideal subjects for studies of basal metabolism,⁹ body composition during euthermic and hibernating states, and gene expression during hibernation.⁸ Other research applications for which ground squirrels are useful include the study of liver neoplasia,^10,15,18 cholesterol gallstones,¹⁷ obesity, and diabetes mellitus.¹⁵Ground squirrels seldom produce fertile matings in captivity, and research colonies must be maintained by capture from the wild, posing biosecurity challenges for such colonies. Despite the need to capture of wild ground squirrels for research, these animals habituate easily and adapt well to captivityKnowledge of the pathology of captive ground squirrels is important to investigators and veterinarians who maintain colonies for research purposes. Although wild ground squirrels are used as a laboratory animal in some research institutions, little information is available in the scientific literature about the naturally occurring diseases and pathology of ground squirrels, with the exception of hepatic tumors.¹⁸ The purpose of the current study was to describe conditions that may occur in a research colony of wild-caught ground squirrels, potentially resulting in death or the need for humane euthanasia. The species included in this study were primarily U. richardsonii and U. columbianus, with 6 C. lateralis.     

物化因子结合诱变选育耐温型法夫酵母

以法夫酵母为出发菌株,采用紫外线及甲基磺酸乙酯对其进行诱变育种,筛选出1株最适生长温度比诱变前提高10℃且虾青素产量可达到5．08mg／L的突变菌株,该菌株经多次传代生产性能稳定。     

Demonstration of Type I Insulin-Like Growth Factor Receptors on Human Platelets

Abstract

Human platelets, freshly isolated from healthy human adults, express receptors for insulin-like growth factor I. The IC₅₀ for displacement of ¹²⁵I-IGF-I binding by unlabeled IGF-I was 0.2 nM, by IGF-II 32 nM and by insulin 160 nM. Scatchard analysis of IGF-I binding demonstrates dissociation constants of 0.14 ± 0.08 nM for high affinity binding site and 54 ± 18 nM for low affinty binding site. The presence of the α-subunit of type I IGF receptor, as high affinity binding site, was verified by affinity crosslinking of ¹²⁵I-IGF-I to platelet surface membranes. Under reducing con-conditions a M_r= 135,000 band was preferentially labeled. The complete type I IGF receptor complex, which revealed under nonreducing conditions, has an approximately molecular mass of M_r > 400,000. The immunoprecipitation of the ¹²⁵I-IGF-I cross-linked type I receptor with αIR-3 confirmed the results achieved by affinity crosslinking.     

表达犬细小病毒VP2蛋白重组犬2型腺病毒的构建及鉴定

对CAV-2的E3区的Ssp I片段进行缺失构建了E3区缺失载体pVAXΔE3,然后将CPV VP2表达盒连接到pVAXΔE3的E3区缺失处,构建了CPV VP2表达盒的转移载体pΔECPV-VP2,用SalI NruI分别对pPoly2-CAV2和pΔECPV-VP2进行双酶切,分别进行琼脂糖凝胶电泳回收目的片段,将含CPV VP2表达盒的SalI NruI片段定向克隆入pPoly2-CAV-2载体,获得了含CPV VP2表达盒重组CAV-2基因组的质粒pCAV-2/CPV-VP2。用AscI ClaI对pCAV-2/CPV-VP2进行双酶切,释放CAV-2/CPV-VP2重组基因组,将CAV-2/CPV-VP2基因组与去除SalI NruI片段的CAV-2基因组的两个片段混合,利用脂质体介导共转染DK细胞,出现病变,获得重组病毒CAV-2/CPV。并且,从形态学、基因组水平、目的基因的转录及重组病毒的生长特征等方面进行了鉴定。结果证明,CAV-2/CPV具有典型的CAV-2形态特征,CAV-2/CPV在繁殖的过程中没有对CPV VP2表达盒片段进行缺失或重排,并且能够转录CPV VP2的mRNA。CAV-2/CPV的繁殖速度比野生型CAV-2的繁殖速度慢。     

Deficiency in a Glutamine-Specific Methyltransferase for Release Factor Causes Mouse Embryonic Lethality

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N⁶-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.Nucleic acids, proteins, carbohydrates, and lipids, as well as a body of small molecules, are subject to methylation in a wide variety of biological contexts (3). The majority of methylation reactions are catalyzed by S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). These enzymes ubiquitously exist in species from all three domains of life.Methylation of DNA occurs on one of two bases: cytosine or adenine (19). In prokaryotes, adenine methylation is as widespread as cytosine methylation. In contrast, eukaryotic genomes are devoid of adenine methylation or this type of methylation is too rare to be detected (23, 26). Intriguingly, two putative N⁶-adenine DNA MTases, named N6amt1 and N6amt2, are encoded in the mouse and human genomes. In addition to the conserved AdoMet-binding signature motif GXGXG and other sequence elements, they possess the NPPY motif characteristic of the N⁶-adenine or N⁴-cytosine DNA MTases in bacteria (6, 14). N6amt1 was thus proposed as an AdoMet-dependent DNA MTase, although no evidence had been provided that N6amt1 could methylate DNA (23).No functional clue for N6amt1 existed until two groups independently identified Escherichia coli HemK, distantly related to N6amt1, as a protein MTase for polypeptide release factors RF1 and RF2 (8, 17). The HemK gene was initially discovered in a genetic screen for heme biosynthesis mutants (18), although subsequent studies revealed no direct involvement in heme metabolism. The presence of an NPPY motif, thought to be restricted to members of the adenine and cytosine amino methyltransferases, led to the suggestion that HemK could be an AdoMet-dependent DNA MTase (2). However, a series of genetic and biochemical experiments finally revealed that HemK methylates the side-chain amide group of a glutamine residue in the universally conserved tripeptide motif GGQ of the two release factors in E. coli (8, 17). Methylation of the release factors ensures efficient translation termination and release of newly synthesized peptide from the ribosome (16). Similarly, the yeast HemK homologue, YDR140w (Mtq2p), was confirmed to methylate the eukaryotic release factor eRF1 on a corresponding glutamine residue (9, 22). More recently, the human homologue N6amt1 (HemK2) was reported to methylate release factor 1 (eRF1) in vitro (5).We initially sought to characterize the function of N6amt1 as a potential DNA adenine MTase. Interestingly, the human N6amt1 gene is located on chromosome 21q21.3, a critical region for Down syndrome (1, 20). In this study, we report the identification of murine N6amt1 as a glutamine-specific MTase of eRF1 both in vitro and in vivo. Mammalian eRF1, the only mammalian release factor, is indeed methylated at the glutamine residue of the GGQ motif. Inactivation of the N6amt1 gene by targeted disruption led to embryonic lethality in the mouse. These data confirm that N6amt1 functions as a protein MTase in mammals and indicate that modulation of the eRF1 activity by N6amt1-mediated glutamine methylation might be essential for embryo viability.     

Factor VII activating protease (FSAP): a novel protease in hemostasis

Recently a novel serine protease in human plasma was described and was named PHBP, PHBSP or factor seven activating protease (FSAP), respectively, the latter according to the finding that it can support coagulation by factor VII activation. Later on FSAP was identified as a potent activator of single chain plasminogen activators, in particular of prourokinase, as well. The physiological role of FSAP is still speculative, but recent studies suggest a contribution to hemostasis. Due to its affinity to glycosaminoglycans a role in cell-associated or extracellular proteolytic events is also likely. The impact of a very recently uncovered frequent polymorphism impairing the prourokinase activation potential of FSAP needs to be investigated in more detail.