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Miscellaneous Medicare and Medicaid amendments--HCFA. Final rule.

These regulations make the following changes in the Medicare and Medicaid rules: 1. Remove from the Medicare rules the lists of deductible and coinsurance amounts that are revised annually. 2. Establish the conditions for Medicare Part B to pay for an antigen that is administered by someone other than the physician who prepares the antigen. 3. Provide that specified equipment is required to be available in an ambulatory surgical center (ASC) only if the medical staff of the ASC considers it necessary. 4. Clarify the rules on agreements with Medicare intermediaries and carriers and on coordination of their activities with the activities of peer review organization (PROs). 5. Correct an involuntary omission by adding "skilled nursing facility services for individuals under 21" to the list of Medicaid services for which Federal financial participation may be continued for up to 30 days after termination of the provider agreement. 6. Revise the rules on denial of Medicare provider agreements to reflect Bankruptcy Code changes under which a provider agreement may not be denied solely because of bankruptcy. These amendments are necessary to simplify, clarify, or conform minor aspects of the Medicare rules on: Deductibles and coinsurance. Payment for antigens. Equipment required in ASCs. Agreements with intermediaries and carriers. Coordination of the activities of intermediaries and carriers and those of PROs. The amendments also correct an omission in the Medicaid rules on Federal financial participation. The first amendment is purely a matter of simplification. The other changes are intended to ensure that users of HCFA regulations have the clear understanding necessary for uniform application.

Centers for Medicare and Medicaid Services, U.S.↗

Comparative evolutionary genomics unveils the molecular mechanism of reassignment of the CTG codon in Candida spp.

Using the (near) complete genome sequences of the yeasts Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we address the evolution of a unique genetic code change, which involves decoding of the standard leucine-CTG codon as serine in Candida spp. By using two complementary comparative genomics approaches, we have been able to shed new light on both the origin of the novel Candida spp. Ser-tRNA(CAG), which has mediated CTG reassignment, and on the evolution of the CTG codon in the genomes of C. albicans, S. cerevisiae, and S. pombe. Sequence analyses of newly identified tRNAs from the C. albicans genome demonstrate that the Ser-tRNA(CAG) is derived from a serine and not a leucine tRNA in the ancestor yeast species and that this codon reassignment occurred approximately 170 million years ago, but the origin of the Ser-tRNA(CAG) is more ancient, implying that the ancestral Leu-tRNA that decoded the CTG codon was lost after the appearance of the Ser-tRNA(CAG). Ambiguous CTG decoding by the Ser-tRNA(CAG) combined with biased AT pressure forced the evolution of CTG into TTR codons and have been major forces driving evolution of the CTN codon family in C. albicans. Remarkably, most of the CTG codons present in extant C. albicans genes are encoded by serine and not leucine codons in homologous S. cerevisiae and S. pombe genes, indicating that a significant number of serine TCN and AGY codons evolved into CTG codons either directly by simultaneous double mutations or indirectly through an intermediary codon. In either case, CTG reassignment had a major impact on the evolution of the coding component of the Candida spp. genome.

Amino Acid Sequence↗

Single-nucleotide polymorphisms of T cell receptor zeta chain in patients with systemic lupus erythematosus.

OBJECTIVE: Signaling molecules from the T cell receptor zeta/Fcepsilon receptor gamma (TCRzeta/FcRgamma) family play a critical role in the function of Fcgamma receptors and the TCR and are located on human chromosome 1, where lupus susceptibility genes are located. This study was undertaken to investigate the possibility of polymorphisms and/or mutations of TCRzeta in systemic lupus erythematosus (SLE). METHODS: We amplified the whole coding region of TCRzeta by reverse transcriptase-polymerase chain reaction (PCR) and directly sequenced the PCR products with a dye primer technique to facilitate heterozygote detection. RESULTS: An alternative splicing form of TCRzeta, with a CAG codon (glutamine) inserted at the splice junction of exons 4 and 5, was found both in SLE and in non-SLE subjects. Both splice isoforms of TCRzeta occurred in human mixed peripheral blood mononuclear cells, natural killer cells, and Jurkat T cells. In TCRzeta, 2 silent and 2 missense mutations were found, but neither coding change occurred in the immunoreceptor tyrosine-activation motif. No unique mutations were found in Caucasian, African American, Hispanic, Chinese, or Japanese SLE patients living in North America. CONCLUSION: The uncommon and equal occurrence of novel single-nucleotide polymorphisms in both SLE patients and normal subjects makes it improbable that they play important roles in genetic susceptibility to SLE.

Alternative Splicing↗

A mutant hook-associated protein (HAP3) facilitates torsionally induced transformations of the flagellar filament of Escherichia coli.

Two mutants with defects in hook-associated protein 3 (HAP3) were isolated that exhibit impaired swimming only when they interact with a solid surface or a semisolid matrix. Motility and chemotaxis were normal in liquid media, even in media containing viscous agents, but cells failed to swarm in 0.28% agar. Mutants appeared to carry a full complement of flagella of normal configuration and length. However, filaments rotating counterclockwise close to a glass surface transformed from normal to straight, while filaments rotating clockwise transformed from curly to straight. Both transformations propagated from base to tip, as expected if torsionally induced. The mutations mapped to the middle of flgL, to structural gene for HAP3, and sequence analysis revealed the same coding change in both mutants: a substitution of cysteine for arginine 168. Our results show that the ability of a filament composed of normal flagellin subunits to resist mechanical stress depends on the structure of the protein (HAP3) to which it is attached at its base. The N-terminal sequence of HAP3 was found to be similar to the N-terminal sequence of flagellin, and the possibility that it provides a nucleation site for the C-terminal region of flagellin is discussed.

Amino Acid Sequence↗

Sequence variation in the capsid protein genes of human papillomavirus type 16 and type 31.

The sequences of the capsid genes of a human papillomavirus type 16 (HPV 16) DNA and an HPV 31 DNA were determined. The HPV 16 DNA contained genes coding for the most variable HPV 16 capsid proteins yet identified (17 variable amino acids). Three of six coding changes in the HPV 31 DNA occurred at positions equivalent to ones where variable amino acids in HPV 16 have been observed. Variable amino acids in both viruses occurred predominantly in regions which showed amino acid variation when closely related types of HPV were compared; thus, most of the factors which determined the intratypic variation in the capsid proteins of the viruses described here were likely the same as those which determined differences between the capsid proteins of different HPV types.

Base Sequence↗

Nucleotide sequences of the neuraminidase genes of influenza A/Leningrad/134/57 (H2N2) virus and two of its live, attenuated, cold-adapted variants.

The nucleotide sequences of the neuraminidase (NA) genes of the A/Leningrad/134/57 (H2N2) wild-type (Len/wt) virus as well as two of its live attenuated, cold-adapted (ca) variants, A/Leningrad/134/17/57 (Len/17) and A/Leningrad/134/47/57 (Len/47), were determined. In comparison with Len/wt, one nucleotide change (C-225 to A) was found in the NA gene of Len/17. This change codes for a Thr-to-Asn substitution at position 69 of NA. The NA gene of the more attenuated Len/47 ca virus has one silent (T-814 to C) and two coding nucleotide substitutions, C-78 to T (Ala-20 to Val) and C-225 to A (Thr-69 to Asn). These sequence data were used to design a PCR-restriction technique to determine the origin of the NA gene in candidate live, attenuated vaccine reassortants made by reassorting these ca strains with current field viruses.

Amino Acid Sequence↗

Novel polymorphisms in HLA-DOA and HLA-DOB in B-cell malignancies.

In B cells, HLA-DO controls HLA-DM-mediated peptide loading on MHC class II molecules. We analyzed whether HLA-DO mutations are associated with autoimmune diseases characterized by an autoantibody component and with a linkage to HLA-DR or HLA-DQ. These diseases include systemic lupus erythematosus, rheumatoid arthritis, celiac disease, and Graves' disease. In addition, several B-cell leukemias were screened for mutations in HLA-DO. A limited number of polymorphisms in DOA and DOB were found, most of which are non-coding changes or result in a conserved amino acid change. A novel non-conserved Arg to Cys mutation in DOA was found in a patient suffering from chronic lymphocytic leukemia. Further analysis did not reveal any effect on the function of HLA-DO. We conclude that HLA-DO variants are not critically involved in the autoimmune diseases and B-cell leukemias studied here.

Autoimmune Diseases↗

Evolutionary conservation and tissue-specific processing of Hoxa 11 antisense transcripts.

We previously described the existence of abundant, processed, polyadenylated murine Hoxa 11 antisense transcripts. Of particular interest, in the developing limbs the antisense transcripts were observed to be present in a pattern complementary to that of the sense transcripts, suggesting a possible regulatory function (Hsieh-Li et al. 1995). We have analyzed the human HOXA 11 genomic locus, showing strong evolutionary conservation of regions potentially encoding antisense transcripts. Human HOXA 11 fetal kidney antisense cDNAs were identified and sequenced, demonstrating the evolutionary conservation of Hoxa 11 antisense transcription. As for the mouse, the human antisense RNAs were polyadenylated and showed several alternative processing patterns, but shared the sequences of a common 3' exon. The evolutionary conservation of the opposite strand transcripts strongly suggests function. A significantly long open reading frame was observed, but mouse-human comparisons argued against true coding function. Murine kidney Hoxa 11 antisense transcription and processing was also examined, revealing tissue-specific differences between limb and kidney. A novel procedure, designated Race in Circles, was devised and used to define mouse limb antisense transcription start sites. Furthermore, comparisons of human, mouse, and chicken sense transcript Hoxa 11 homeobox nucleotide sequences and their respective encoded homeodomains indicate a very strong selective pressure in vertebrates against mutations that result in coding changes. Given the significant differences in amino acid sequences of the homeodomains of different Hox genes, this observation argues for individual homeodomain functional specificity.

5' Untranslated Regions↗

Transmembrane segment 10 is important for substrate recognition in Ga12 and Hxt2 sugar transporters in the yeast Saccharomyces cerevisiae.

A systematic series of chimeras between Ga12 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOH-terminus that has been previously identified as the critical substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the galactose transport-active chimeras were found to possess Ga12-derived transmembrane segment (TM) 10. Of the 35 amino acids in the TM1O region, only 12 differ between Ga12 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.

Amino Acid Sequence↗

Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein.

Experiments with monoclonal antibodies raised against two laboratory strains of Sindbis virus, SB and SIN, suggested the existence of a strain-specific neutralizing antigenic site (E2-b) on the E2 glycoprotein. A comparison of monoclonal antibody binding patterns and E2 glycoprotein gene sequences of six laboratory strains distinguished three different configurations of E2-b that correlated with specific amino acid substitutions at position 216 of the E2 glycoprotein. Further study of neutralization escape mutants selected with E2-b-specific antibodies confirmed that amino acid 216 is a major determinant of the E2-b antigenic site. Eight of nine mutants showed a coding change at position 216. One neutralization escape mutation created a new glycosylation site at position 213 and resulted in an E2 protein with an altered migration rate in SDS-PAGE. The neutralization escape mutants studied included amino acid substitutions not found in the laboratory strains that revealed differing binding requirements for two E2-b-specific monoclonal antibodies. The E2-b site is contrasted with the E2-c neutralizing antigenic site described previously (R.A. Olmsted, W.J. Meyer, and R.E. Johnston, 1986, Virology 148, 245-254).

Amino Acid Sequence↗

Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses.

Canine parvovirus (CPV) is a recently recognized pathogen of dogs that is similar to the long-recognized feline, mink, and raccoon parvoviruses. Relationships between the viruses determined from DNA sequences of the capsid protein genes of 10 virus isolates showed the CPV isolates to be closely related to the other viruses, although comprising a distinct group. No immediate ancestor of CPV was observed amongst the mink, cat, or raccoon viruses examined. Three different directly repeated sequences were present within the noncoding region downstream from the capsid protein genes. Analysis of recombinants between CPV and feline panleukopenia virus at restriction sites within the capsid protein genes mapped a CPV-specific neutralization epitope on the virus capsid, differences in the pH dependence of hemagglutination, and part of the determinant of canine host range between 59 and 64 genome map units (m.u.). Those differences were therefore the result of up to three nucleotide or predicted amino acid sequence differences in that region. A second region between 64 and 73 m.u., which may affect the viability of certain recombinant viruses, contained four nucleotide differences, one of which was a coding change.

Animals↗

Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones.

DNA sequences between 0 and 98.8 genome map units (m.u.) from canine parvovirus (CPV) and feline panleukopenia virus (FPV) were cloned into plasmid vectors to form infectious molecular clones. Those plasmids were transfected into permissive cells and viruses recovered were shown to contain intact genomes, having regenerated the complete viral 5' ends up to 100 m.u. The viruses derived from the plasmids were compared to the original viruses, and shown to be indistinguishable in antigenic type, hemagglutination (HA) type and host range. The plasmid origin of the viruses was shown by preparing recombinant clones between CPV and FPV, and demonstrating the recombinant nature of the resulting viruses by restriction mapping and by sequencing viral DNA across the recombination sites. The sequences of our wild-type isolates CPV-d and FPV-b were completed, revealing 50 nucleotide sequence differences, of which 16 determined coding changes--5 in NS-1,2 in NS-2, and 9 in VP-2 protein. The sequences of the 5' ends (95.3-100 m.u.) of both viruses were also determined. Analysis of recombinant viruses mapped both CPV- and FPV-specific antigenic epitopes, the pH dependence of HA, and sequences affecting canine host range of the viruses within the VP-1 and VP-2 structural protein genes. Most of the specific changes were shown to be either on, or within one amino acid of, the surface of the virus capsid, indicating that the exposed surface of the parvovirus capsid plays an important role in determining a number of virus functions. The specific epitopes were affected by differences in a raised area on the capsid ("threefold spike"), while the pH dependence of HA difference was adjacent to a depression in the surface of the capsid at the twofold axis of symmetry.

Animals↗

Molecular characterization and expression of glycoprotein gene of Hantavirus R22 strain isolated from Rattus norvegicus in China.

A cDNA containing the complete open reading frame of the M genome segment of Hantavirus R22 strain isolated from Rattus norvegicus in China, was amplified by polymerase chain reaction (PCR), and then cloned. The M segment is 3656 nucleotides in length with a predicted region of 3402 bases encoding a precursor glycoprotein of 1134 amino acids subsequently processed into viral glycoproteins 1 and 2 (G1 and G2). A strain comparison between R22 and SR11 (isolated from a rat in Japan), and Hantaan 76-118 (isolated from Apodemus in Korea), and Hallnas B1 (isolated from a bank vole in Sweden) revealed 95%, 74%, and 53% homologies at the deduced amino acid sequence level respectively. This suggests that the rodent host species may be a more important determinant of genetic relationships than geographic proximity. Six potential asparagine linked glycosylation sites (five in G1 and one in G2) were identified, and among them all are conserved in SR11, five in Hantaan virus and four in Hallnas B1 virus. Although different degrees of homology exist among these four viruses at amino acid sequence level, more than 90% of the cysteine residues are conserved, suggesting that structural homology may be very strong between the Hantaviruses. Genetic differences in the M segment genome of R22 and SR11 viruses, within the same serotype viruses, were found as random coding changes; some limited to single amino acids, others in clusters. A recombinant vaccinia virus that contained the fully activated M segment cDNA of R22 was constructed. This recombinant virus expressed two glycoproteins G1 and G2 identical to R22 virus G1 and G2 in molecular weight, cleavage pattern and cellular immunofluorescent patterns.

Amino Acid Sequence↗

Chemical methods of protein synthesis and modification.

Chemical and recombinant methods have continued to complement one another in the synthesis of protein analogues. Chemical methods remain particularly valuable when non-coded modifications are to be introduced, although it has been accepted since the commercialization of semisynthetic human insulin that they can also be used effectively for coded changes, in certain cases. The main objective of all such operations is not methodological, but is the production of molecules for practical use and further study. This goal has been reached frequently by chemical means during the past year.

Base Sequence↗

Front-line police officers' practices, perceptions and attitudes about the enforcement of impaired driving laws in Canada.

A survey of front line police officers' practices, perceptions and attitudes regarding detection of impaired driving, processing of driving while impaired (DWI) charges, criminal court proceedings and DWI sanctions was conducted across Canada. A sample of 1545 officers of all jurisdictions across the country, representative of different types of police services (i.e. national, provincial, municipal) and types of policing (i.e. traffic, general duty) were surveyed by mail. The results, based on a 71% response rate, indicate that: an average of 7.5 charges/year are laid by officers resulting mainly from erratic driving; videotaping and mobile breath testing could improve efficiency of DWI enforcement; it takes an average of 2 h 48 min to process each DWI charge; about 2/3 of officers say plea bargaining occurs at least sometimes; the average length of DWI trial is over 4 h; less than half of officers think Crown Attorneys are adequately prepared for DWI cases; about 3/4 of officers think the accused escapes conviction on a legal technicality at least sometimes; about 30% of officers say short-term licence suspensions and other forms of discretion are used at least sometimes; DWI places fifth in priority among 15 offences, up from eighth in a 1981 survey; DWI is a priority for most police management but human resources are not adequate; and there is greater support for administrative than for Criminal Code changes. Multiple regressions indicated that the number of DWI charges laid by officers depended mainly on the officers' personal priority regarding DWI enforcement. The results suggest that many officers want to enforce DWI laws but that the numerous procedural and legal barriers that they confront often force them to exercise discretion in the laying of DWI charges.

Accidents, Traffic↗

Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein.

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer is associated with mismatch repair deficiency. Most predisposing mutations prevent the production of functional mismatch repair protein. Thus, when the wild-type copy is also inactivated, the cell becomes mismatch repair deficient, and this leads to a high degree of microsatellite instability in tumors. However, tumors linked to nontruncating mutations may display positive or partly positive immunohistochemical staining of the mutated protein and low or atypical microsatellite instability status, which suggests impaired functional activity but not a total lack of mismatch repair. We found human mutL homology (hMLH) 1 del616, one of the most widespread recurring mutations in hereditary nonpolyposis colorectal cancer, segregating in a large hereditary nonpolyposis colorectal cancer family. Because the predicted coding change is a deletion of only 1 amino acid, the pathogenicity of the mutation was evaluated. METHODS: Many analyses were performed to assess the pathogenicity of hMLH1 del616 and to study the expression and function of the mutated messenger RNA and protein. RESULTS: Genetic and immunohistochemical evidence supported hMLH1-linked cancer predisposition in this family. Microsatellite instability varied from low to high, and the hMLH1 protein was lost in 2 tumors but was partly detectable in 1 tumor. Whereas similar optimal amounts of mutated hMLH1 del616 and wild-type hMLH1 proteins were equally functional in an in vitro mismatch repair assay, the amount of in vivo-expressed hMLH1 del616 was much lower than the amount of wild-type protein; this suggests that the deletion imparts instability to the mutant protein. CONCLUSIONS: Our results suggest that the pathogenicity of hMLH1 del616 is not linked to nonfunctionality, but to shortage of the functional protein.

Adaptor Proteins, Signal Transducing↗

High-affinity ivermectin binding to recombinant subunits of the Haemonchus contortus glutamate-gated chloride channel.

Glutamate-gated chloride channels (GluCls) are targets for the avermectin anthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown that two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluClbeta, Hcgbr-2A and Hcgbr-2B) with high amino-acid identity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluClalpha, from H. contortus eggs. Sequence comparisons suggested that this subunit was closely related to, but not orthologous with, the C. elegans GluClalpha1, alpha2 or alpha3/GBR-2 subunits ( approximately 55% amino-acid identity). The HcGluClalpha cDNA from an ivermectin-resistant isolate contained no coding changes from the wild-type. All of the known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by functional data from the C. elegans orthologues, the Hcgbr-2A and HcGluClbeta subunits failed to bind [3H]ivermectin. The Hcgbr-2B and HcGluClalpha subunits bound [3H]ivermectin with high affinity; the K(d) values were 70+/-16 and 26+/-12 pM, respectively. This binding was inhibited by a variety of avermectins, though cold ivermectin was the most potent inhibitor of [3H] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all failed to compete for ivermectin binding to either subunit. The affinity of [3H]ivermectin binding to H. contortus L3 P2 larval membrane preparations was re-examined and found to be 70+/-7 pM. The properties of orthologous GluCl subunits are likely to be conserved across species, but the repertoire and relative importance of those subunits may vary.

Animals↗

Genetic analysis of canine parvovirus isolates (CPV-2) from dogs in Italy.

Genetic and antigenic properties of 62 field isolates of canine parvovirus (CPV-2) collected from 1994 to 2001 in Italy were investigated. Antigenic characterisation was conducted using specific monoclonal antibodies (Mabs). The VP1\VP2 gene was amplified by PCR and characterised with restriction endonucleases to detect the 297 and 265 variant. The VP2 gene of 16 isolates was sequenced and molecular genetic analysis was conducted. The antigenic type prevalent among our isolates is type 2a as well as the 297 variant, which is also prevalent in the rest of Europe. Only the 9.7% of the isolates have the T265P mutation. The VP2 sequences of CPV-2 isolates were very similar to recent Asian isolates. In the threefold spike of CPV-699 a coding change was detected in the 440 residue where threonine was substituted by alanine: the same mutation has been found in two Asian CPV-2 isolates from leopard cats [Virology 278 (2000) 13]. Phylogenetic analysis revealed that the Italian CPV-2 strains followed the same evolution as observed in other countries and they gave no indication of a separate lineage.

Animals↗