Isolation and identification of adenovirus from conjunctival scrapings over a two-year period (between 2001 and 2003) in Yokohama, Japan

Over a 2-year period between 2001 and 2003, a total of 115 conjunctival scrapings were collected from patients with keratoconjuctivitis from several hospitals in Yokohama, Japan. Out of 115, 94 (82.4%) cases of adenoviruses were detected by polymerase chain reaction (PCR); 60 (52.1%) by cell culture isolation; and 16 (14.0%) by enzyme-linked immunosorbent assay (ELISA). The serotypes were determined by PCR- restriction fragment length polymorphism analysis (PCR-RFLP) and by the neutralization test (NT). PCR-RFLP was performed using a combination of endonucleases such as HhaI, AluI, and HaeIII. Of the 94 PCR-positive samples, the serotypes of 91 (96.8%) were identified by PCR-RFLP analysis (adenovirus 3: 50%, 4: 11%, and 8: 32%). Out of the 115 samples, 60 samples were identified by the neutralization (adenovirus 3, 4, 7, and 8). When both PCR-RFLP and the neutralization techniques were used, 53.2%, 11.7%, 1.1%, and 34% of the samples were identified as adenovirus 3, 4, 7, and 8, respectively. In contrast to the results of a nationwide surveillance report, adenovirus 3 was found as a major cause of keratoconjunctivitis in the Yokohama area. The nationwide surveillance report did not reflect accurately the epidemiological situation in the local area. In order to obtain surveillance data that would be useful for the prevention of an adenovirus conjunctivitis epidemic, it seems that local epidemiology is more important than that nationwide surveillance.     

Human T‐lymphotropic virus type 1 (HTLV‐1) genetic typing in Kakeroma Island,an island at the crossroads of the ryukyuans and Wajin in Japan,providing further insights into the origin of the virus in Japan

Peripheral blood samples were collected from 23 human T‐lymphotropic virus type‐1 (HTLV‐1) carriers residing in Kakeroma Island, Japan (Kagoshima Prefecture, Oshima County, Setouchi Town), one of the most highly endemic areas in Japan. The samples were subjected to amplification by PCR and sequencing of the Long Terminal Repeat in order to reconstruct a phylogenetic tree of HTLV‐1 isolates. Restriction Fragment Length Polymorphism (RFLP) analysis of env region was also conducted for subgrouping of HTLV‐1. Although one sample could not be amplified by PCR, and three more could not be sequenced due to the existence of conspicuous nonspecific bands or repeated sequences, the phylogenetic analysis revealed that the remaining 19 isolates obtained from Kakeroma Island belonged to either the Transcontinental or the Japanese subgroups of the Cosmopolitan subtype, one of the three major subtypes. The RFLP data corresponded closely with the typing data throughout the sequencing. The proportion of the Transcontinental subgroup among the isolates was 26.3% (5 of 19) by sequence analysis and 27.3% (6 of 22) by RFLP. Unlike in Taiwan, China and Okinawa, the Japanese subgroup was dominant in Kakeroma Island. The analysis would also suggest that the Japanese subgroup seems not to have derived from the Transcontinental subgroup, but rather that the Transcontinental subgroup came to Japan first and was followed later by the Japanese one. J. Med. Virol. 81:1450–1456, 2009. © 2009 Wiley‐Liss, Inc.     

Determination of HIV-1 subtypes (A-D, F, G, CRF01_AE) by PCR in the transmembrane region (gp41) with novel primers

HIV-1 has a huge genetic diversity. So far, nine subtypes have been isolated, namely, subtypes A, B, C, D, F, G, H, J, and K. Epidemiological study provides information which may help in the development of HIV-1 prevention programs or health policies. In the future, subtyping may also be critical for vaccine development, and an effective anti-viral drug will need to be effective for different subtypes of HIV virus. The analysis of the nucleotide sequence of the v3 region is considered the most reliable method for determining the HIV-1 subtype. However, the procedures for determining the v3 sequences are complicated and time consuming, requiring expensive reagents, equipment, and well-trained personnel. The polymerase chain reaction (PCR) method using subtype-specific primers for HIV-1 subtyping is easier and faster. The objective of this study was to develop subtype-specific primers for subtyping PCR. The specific primers were designed for subtypes A, B, C, D, F, G, and CRF01_AE, and these primers could be applied to assay for various HIV-1 subtypes in the clinical samples. The specific primers were designed for each subtypes in the gp41 region. The result of PCR was compared with the subtypes which was determined by the v3 sequence. The results of subtyping by PCR using the newly designed primers could detect 29 of 33 patients tested, and all matched those obtained by nucleotide sequencing of the env v3 region except for three subjects, which were differentiated as CRF02_AG. The newly designed primers functioned accurately and conclusively. In comparison with PCR as a method for the determination of subtypes, sequence analysis requires better-trained personnel, more expensive reagents, and more equipment and time.     

湖北汉族人群C4限制性片段长度多态性的研究

用限制性内切酶ＴａｑＩ和Ｃ４基因５￣＇末端探针ｐＡＴ－Ａ检测５８份湖北地区健康献血员基因组ＤＮＡ的Ｃ４基因限制性片段长度多态性（ＲＦＬＰ），观察到７．０ｋｂ、６．０ｋｂ和５．４ｋｂ三种Ｃ４基因片段，它们共组成三种常见的Ｃ４基因ＲＦＬＰ表型，即Ａ：７．０５－４ｋｂ（频率为２４．１４％））；Ｂ：７．０－６．０ｋｂ（频率为２０．６９％）和Ｃ：７．０－６．０－５．４ｋｂ（频率为５５．１７％，在所检测的样本中未观察到６．４ｋｂ的Ｃ４基因片段。     

Neither molecular diversity of the envelope,immunosuppression status,nor proviral load causes indeterminate HTLV western blot profiles in samples from human T‐cell lymphotropic virus type 2 (HTLV‐2)‐infected individuals

Although human T‐cell lymphotropic virus type 2 (HTLV‐2) is considered of low pathogenicity, serological diagnosis is important for counseling and monitoring. The confirmatory tests most used are Western blot (WB) and PCR. However, in high‐risk populations, about 50% of the indeterminate WB were HTLV‐2 positives by PCR. The insensitivity of the WB might be due to the use of recombinant proteins of strains that do not circulate in our country. Another possibility may be a high level of immunosuppression, which could lead to low production of virus, resulting in low stimulation of antibody. We found one mutation, proline to serine in the envelope region in the position 184, presented at least 1/3 of the samples, independent the indeterminate WB profile. In conclusion, we found no correlation of immune state, HTLV‐2 proviral load, or env diversity in the K55 region and WB indeterminate results. We believe that the only WB kit available in the market is probably more accurate to detect HTLV‐1 antibodies, and some improvement for HTLV‐2 detection should be done in the future, especially among high‐risk population. J. Med. Virol. 82: 837–842, 2010. © 2010 Wiley‐Liss, Inc.     

Large deletions of the APC gene in 15% of mutation-negative patients with classical polyposis (FAP): a Belgian study

The now-classical model of replication slippage can in principle account for both simple deletions and tandem duplications associated with short direct repeats. Invariably, a single replication slippage event is invoked, irrespective of whether simple deletions or tandem duplications are involved. However, we recently identified three complex duplicational insertions that could also be accounted for by a model of serial replication slippage. We postulate that a sizeable proportion of hitherto inexplicable complex gene rearrangements may be explained by such a model. To test this idea, and to assess the generality of our initial findings, a number of complex gene rearrangements were selected from the Human Gene Mutation Database (HGMD). Some 95% (20/21) of these mutations were found to be explicable by twin or multiple rounds of replication slippage, the sole exception being a double deletion in the F9 gene that is associated with DNA sequences that appear capable of adopting non-B conformations. Of the 20 complex gene rearrangements, 19 (seven simple double deletions, one triple deletion, two double mutational events comprising a simple deletion and a simple insertion, six simple indels that may constitute a novel and non-canonical class of gene conversion, and three complex indels) were compatible with the model of serial replication slippage in cis; the remaining indel in the MECP2 gene, however, appears to have arisen via interchromosomal replication slippage in trans. Our postulate that serial replication slippage may account for a variety of complex gene rearrangements has therefore received broad support from the study of the above diverse series of mutations.     

A novel frameshift mutation (141delT) in exon 1 of the 21‐hydroxylase gene (CYP21) in a patient with the salt wasting form of congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disease with a wide range of clinical manifestation. In 90‐95% of the cases it is caused by 21‐hydroxylase deficiency (OMIM #201910) due to mutations of the CYP21 gene (GDB Accession #M12792). In most cases the CYP21‐inactivating point mutations are transferred by apparent gene conversions from CYP21P to CYP21. In only a few cases point mutations have been described, which are not present in the pseudogene. Using Southern blot analysis and DNA sequencing we have identified a novel mutation (141delT) of the CYP21 gene in a patient suffering from the salt wasting form of CAH. This results in a premature termination of a truncated protein at amino acid position 51 (L51X), which is likely to result in an enzyme with no activity. This novel mutation has not been reported to occur in the CYP21P alleles and it was not found in the CYP21P alleles in this CAH family. Hum Mutat 14:90–91, 1999. © 1999 Wiley‐Liss, Inc.     

Microbial competition, lack in macronutrients, and acidity as main obstacles to the transfer of basidiomycetous ground fungi into (organically or heavy-metal contaminated) soils

Non-symbiotic soil microorganisms which have been expensively engineered or selected to support plant nutrition, control root diseases, degrade xenobiotic hydrocarbons, and repress or stimulate heavy metal uptake of plants fail to survive in target soils. This prompted studies into the role of chemistry and microbial pre-colonization of 23 top soils in long-term growth of basidiomycetes. Fungi are seen as auxiliary agents in soil remediation. Untreated soils (1.5 L) were colonized by lignocellulose preferring ground fungi such as Agaricus aestivalis, A. bisporus, A. campestris, A. edulis, A. macrocarpus, A. porphyrizon, Agrocybe dura, A. praecox, Clitocybe sp., Coprinus comatus, Lepista nuda, L. sordida, Macrolepiota excoriata, M. procera, Stropharia coronilla, and S. rugoso-annulata. Spawn mycelia of fairy-ring-type fungi such as Agaricus arvensis, A. fissuratus, A. langei, A. lanipes, A. pilatianus, Lyophyllum sp., and Marasmius oreades died back in contact with non-sterile soils. Fungal growth correlated positively with the soils' Ct Ca K Mg content and negatively with microbial CO2 evolution. Pasteurization and autoclaving increased mycelial growth and life span in soils pH 6.6-8.2. Growth of pH-sensitive but not of pH-tolerant fungi was inhibited on the Ca-deficient soils pH 4-4.4 (-5.6) and was not improved by autoclaving. The pretended fungistasis of acid soils to pH-sensitive fungi was controlled by N P K mineral (pH not altering) or organic (pH increasing) fertilizing as well as by neutralization with NaOH or CaCO3. Although microbial competition was mortal to 33% of the fungal mycelia inserted into natural unplanted soils, further seriously antifungal effects beyond those pretended by low pH conditions and shortage in mineral macronutrients were not identified.     

Use of a generic polymerase chain reaction assay detecting human T-lymphotropic virus (HTLV) types I,II and divergent simian strains in the evaluation of individuals with indeterminate HTLV serology

In countries with a low prevalence of human T-lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T-lymphotropic virus (STLV), which give an HTLV-indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T-cell lymphotropic virus strains (PTLV), including the two new types STLV-PH969 and STLV-PP1664, we developed generic and type-specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross-reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross-reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV-IPCR positive and 1 was HTLV-II PCR positive. Sequencing of one of the HTLV-I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV-I. The majority of the seroindeterminate samples (91%) were however HTLV-PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV-I or HTLV-II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples. J. Med. Virol. 52:1–7, 1997.© 1997 Wiley-Liss, Inc.     

Neuronal and glial cell types revealed by NADPH-diaphorase histochemistry in the retina of a teleost fish, the grass goby ( Zosterisessor ophiocephalus , Perciformes, Gobiidae)

The grass goby is a mud-burrowing fish with a rich retinal vasculature appropriate to its hypoxic habitat. NADPH-diaphorase histochemistry was performed on retinal sections and wholemounts to reveal cells that contain nitric oxide synthase and so may be presumed to synthesise nitric oxide, a gaseous intercellular messenger with many roles including vasodilation. Structures that were consistently stained by this method included cone ellipsoids, horizontal cells, Müller cells and their processes, large displaced ganglion cells in the inner nuclear layer (identified by their axons), large interstitial ganglion cells in the inner plexiform layer, and capillary endothelial cells. In wholemounts, horizontal cells were seen to form a regular pattern, contacting each other at their dendritic terminals. Some cells in the ganglion cell layer were weakly stained, but stained bipolar and amacrine cells were not seen. The diaphorase-positive large ganglion cells all formed large, sparsely branched dendritic trees, arborizing near the scleral border of the inner plexiform layer. The displaced and interstitial cells seemed to belong to distinct morphological types, the interstitial cells having smaller somata and trees. Analysis of their spatial distributions in one representative retina confirmed this: the displaced cells formed a highly regular mosaic with a mean spacing (nearest-neighbour distance) of 303 μm, whereas the interstitial cells formed a separate mosaic, almost as regular but with a smaller mean spacing of 193 μm, rising to 217 μm in a sample that excluded the area retinae temporalis. Spatial correlogram analysis showed that these two mosaics were spatially independent. Nitric oxide probably has many roles in the retina. The presence of its synthetic enzyme in Müller cells, which communicate with retinal blood vessels, is consistent with a role in the control of retinal blood flow. Its function in large, mosaic-forming retinal ganglion cells is unknown. Accepted: 29 April 1999     

Pathogenic effects of novel mutations in the P-type ATPase ATP13A2 (PARK9) causing Kufor-Rakeb syndrome, a form of early-onset parkinsonism

Kufor-Rakeb syndrome (KRS) is a rare form of autosomal recessive juvenile or early-onset, levodopa responsive parkinsonism and has been associated with mutations in ATP13A2(also known as PARK9), a lysosomal type 5 P-type ATPase. Recently, we identified novel compound heterozygous mutations, c.3176T>G (p.L1059R) and c.3253delC (p.L1085WfsX1088) in ATP13A2 of two siblings affected with KRS. When overexpressed, wild-type ATP13A2 localized to Lysotracker-positive and LAMP2-positive lysosomes while both truncating and missense mutated ATP13A2 were retained in the endoplasmic reticulum (ER). Both mutant proteins were degraded by the proteasomal but not the lysosomal pathways. In addition, ATP13A2 mRNA with c.3253delC was degraded by nonsense-mediated mRNA decay (NMD), which was protected by cycloheximide treatment. To validate our findings in a biologically relevant setting, we used patient-derived human olfactory neurosphere cultures and fibroblasts and demonstrated persistent ER stress by detecting upregulation of unfolded protein response-related genes in the patient-derived cells. We also confirmed NMD degraded ATP13A2 c.3253delC mRNA in the cells. These findings indicate that these novel ATP13A2 mutations are indeed pathogenic and support the notion that mislocalization of the mutant ATP13A2, resultant ER stress, alterations in the proteasomal pathways and premature degradation of mutant ATP13A2 mRNA contribute to the aetiology of KRS.     

Depletion of the lipid raft constituents, sphingomyelin and ganglioside, decreases serotonin binding at human 5-HT7(a) receptors in HeLa cells

Aim: The localization and function of several G protein‐coupled receptors, including β‐adrenergic receptors and NK 1 receptors, are regulated via lipid rafts in the plasma membrane. These domains are enriched in cholesterol, gangliosides and sphingolipids, and play an important role in regulating signal transduction in most cell types. Serotonin (5‐hydroxytryptamine, 5‐HT), acting via 14 different receptors, regulates as diverse effects as mood, metabolism and smooth muscle contraction. 5‐HT₇ receptors are involved in the regulation of depression, circadian rhythms, thermoregulation and vasodilatation. Ligand binding and signalling via the 5‐HT₇ receptor are regulated by membranous cholesterol. Here we investigated the role of sphingomyelin and gangliosides on binding of 5‐HT to 5‐HT₇ receptors to further examine the role of lipid raft constituents on 5‐HT₇ receptor function. Methods: HeLa cells stably transfected with the human 5‐HT₇ receptor were treated with Fumonisin B₁ or (±)‐threo‐1‐Phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) to reduce sphingomyelin or ganglioside levels, respectively. The effects of these treatments were investigated by the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl‐tetrazolium bromide (MTT) viability assay, cholesterol analysis and [³H]5‐HT binding studies on intact cells. Results: Treatments with 20 μm Fumonisin B₁ for 24 h or with 10 μm PDMP for 48 h had no effects of total levels if 5‐HT₇ receptors, but caused significant decreases in maximum [³H]5‐HT binding to 5‐HT₇ receptors. The effects were cholesterol‐independent as levels of cholesterol remained unaffected by either treatment. Conclusion: These data demonstrate a role for sphingomyelin and gangliosides in regulating binding of [³H]5‐HT to 5‐HT₇ receptors. These observations further strengthen that actions of 5‐HT via 5‐HT₇ receptors are dependent upon lipid raft integrity.     

Congenital insensitivity to pain with anhidrosis (CIPA): Novel mutations of the TRKA (NTRK1) gene,a putative uniparental disomy,and a linkage of the mutant TRKA and PKLR genes in a family with CIPA and pyruvate kinase deficiency

Congenital insensitivity to pain with anhidrosis is an autosomal recessive hereditary disorder characterized by recurrent episodic fever, anhidrosis (inability to sweat), absence of reaction to noxious stimuli, self‐mutilating behavior, and mental retardation. The human TRKA gene (NTRK1), located on chromosome 1q21‐q22 encodes the receptor tyrosine kinase for nerve growth factor. We reported that TRKA is the gene responsible for CIPA and we developed a comprehensive strategy to screen for TRKA mutations and polymorphisms, as based on the gene’s structure and organization. Here we report eight novel mutations detected as either a homozygous or heterozygous state in nine CIPA families from five countries. Mendelian inheritance of the mutations was confirmed in seven families for which samples from either parent were available. However, non‐mendelian inheritance seems likely for the family when only samples from the mother and siblings, (but not from the father) were available. A paternal uniparental disomy for chromosome 1 is likely to be the cause of reduction to homozygosity of the TRKA gene mutation in this family. Interestingly, a Hispanic patient from the USA has two autosomal genetic disorders, CIPA and pyruvate kinase deficiency, whose genetic loci are both mapped to a closely linked chromosomal region. A splice mutation and a missense mutation were detected in the TRKA and PKLR genes from the homozygous proband, respectively. Thus, concomitant occurrence of two disorders is ascribed to a combination of two separate mutant genes, not a contiguous gene syndrome. This finding suggests a mechanism responsible for two autosomal genetic disorders in one patient. All these data further support findings that TRKA defects can cause CIPA in various ethnic groups. This will aid in diagnosis and genetic counseling of this painless but severe genetic disorder. Hum Mutat 18:308–318, 2001. © 2001 Wiley‐Liss, Inc.     

Masticatory muscle architecture in a water‐rat from Australasia (Murinae,Hydromys) and its implication for the evolution of carnivory in rodents

Murines are well known for their generalist diet, but several of them display specializations towards a carnivorous diet such as the amphibious Indo‐Pacific water‐rats. Despite the fact that carnivory evolved repeatedly in this group, few studies have investigated associated changes in jaw muscle anatomy and biomechanics. Here, we describe the jaw muscles and cranial anatomy of a carnivorous water‐rat, Hydromys chrysogaster. The architecture of the jaw musculature of six specimens captured both on Obi and Papua were studied and described using dissections. We identified the origin and insertions of the jaw muscles, and quantified muscle mass, fiber length, physiological cross‐sectional area, and muscle vectors for each muscle. Using a biomechanical model, we estimated maximum incisor and molar bite force at different gape angles. Finally, we conducted a 2D geometric morphometric analyses to compare jaw shape, mechanical potential, and diversity in lever‐arm ratios for a set of 238 specimens, representative of Australo‐Papuan carnivorous and omnivorous murids. Our study reveals major changes in the muscle proportions among Hydromys and its omnivorous close relative, Melomys. Hydromys was found to have large superficial masseter and temporalis muscles as well as a reduced deep masseter and zygomatico‐mandibularis, highlighting major functional divergence among omnivorous and carnivorous murines. Changes in these muscles are also accompanied by changes in jaw shape and the lines of action of the muscles. A more vertically oriented masseter, reduced masseteric muscles, as well as an elongated jaw with proodont lower incisors are key features indicative of a reduced propalinality in carnivorous Hydromys. Differences in the fiber length of the masseteric muscles were also detected between Hydromys and Melomys, which highlight potential adaptations to a wide gape in Hydromys, allowing it to prey on larger animals. Using a biomechanical model, we inferred a greater bite force in Hydromys than in Melomys, implying a functional shift between omnivory and carnivory. However, Melomys has an unexpected greater bite force at large gape compared with Hydromys. Compared with omnivorous Melomys, Hydromys have a very distinctive low mandible with a well‐developed coronoid process, and a reduced angular process that projects posteriorly to the ascending rami. This jaw shape, along with our mechanical potential and jaw lever ratio estimates, suggests that Hydromys has a faster jaw closing at the incisor, with a higher bite force at the level of the molars. The narrowing of the Hydromys jaw explains this higher lever advantage at the molars, which constitutes a good compromise between a wide gape, a reduced anterior masseteric mass, and long fiber lengths. Lever arms of the superficial and deep masseter are less favourable to force output of the mandible in Hydromys but more favourable to speed. Compared with the small input lever arm defined between the condyle and the angular process, the relatively longer mandible of Hydromys increases the speed at the expense of the output force. This unique combination of morphological features of the masticatory apparatus possibly has permitted Hydromys to become a highly successful amphibious predator in the Indo‐Pacific region.