Intraocular Pressure Rise in Subjects with and without Glaucoma during Four Common Yoga Positions

Purpose

To measure changes in intraocular pressure (IOP) in association with yoga exercises with a head-down position.

Methods

The single Center, prospective, observational study included 10 subjects with primary open-angle glaucoma and 10 normal individuals, who performed the yoga exercises of Adho Mukha Svanasana, Uttanasana, Halasana and Viparita Karani for two minutes each. IOP was measured by pneumatonometry at baseline and during and after the exercises.

Results

All yoga poses were associated with a significant (P<0.01) rise in IOP within one minute after assuming the yoga position. The highest IOP increase (P<0.01) was measured in the Adho Mukha Svanasana position (IOP increase from 17±3.2 mmHg to 28±3.8 mmHg in glaucoma patients; from 17±2.8 mmHg to 29±3.9 mmHg in normal individuals), followed by the Uttanasana position (17±3.9 mmHg to 27±3.4 mmHg (glaucoma patients) and from 18±2.5 mmHg to 26±3.6 mmHg normal individuals)), the Halasana position (18±2.8 mmHg to 24±3.5 mmHg (glaucoma patients); 18±2.7 mmHg to 22±3.4 mmHg (normal individuals)), and finally the Viparita Kirani position (17±4 mmHg to 21±3.6 mmHg (glaucoma patients); 17±2.8 to 21±2.4 mmHg (normal individuals)). IOP dropped back to baseline values within two minutes after returning to a sitting position. Overall, IOP rise was not significantly different between glaucoma and normal subjects (P = 0.813), all though glaucoma eyes tended to have measurements 2 mm Hg higher on average.

Conclusions

Yoga exercises with head-down positions were associated with a rapid rise in IOP in glaucoma and healthy eyes. IOP returned to baseline values within 2 minutes. Future studies are warranted addressing whether yoga exercise associated IOP changes are associated with similar changes in cerebrospinal fluid pressure and whether they increase the risk of glaucoma progression.

Trial Registration

ClinicalTrials.gov #{"type":"clinical-trial","attrs":{"text":"NCT01915680","term_id":"NCT01915680"}}NCT01915680     

Chromosome heteromorphism quantified by high-resolution bivariate flow karyotyping.

Maternal and paternal homologues of many chromosome types can be differentiated on the basis of their peak position in Hoechst 33258 versus chromomycin A3 bivariate flow karyotypes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of the same type. Significant peak-position differences between homologues were observed for an average of four chromosome types in each of the karyotypes of 98 different individuals. The frequency of individuals with differences in homologue peak positions varied among chromosome types: e.g., chromosome 15, 61%; chromosome 3, 4%. Flow karyotypes of 33 unrelated individuals were compared to determine the range of peak position among normal chromosomes. Chromosomes Y, 21, 22, 15, 16, 13, 14, and 19 were most heteromorphic, and chromosomes 2-8 and X were least heteromorphic. The largest chromosome 21 was 45% larger than the smallest 21 chromosome observed. The base composition of the variable regions differed among chromosome types. DNA contents of chromosome variants determined from flow karyotypes were closely correlated to measurements of DNA content made of gallocyanin chrome alum-stained metaphase chromosomes on slides. Fluorescence in situ hybridization with chromosome-specific repetitive sequences indicated that variability in their copy number is partly responsible for peak-position variability in some chromosomes. Heteromorphic chromosomes are identified for which parental flow karyotype information will be essential if de novo rearrangements resulting in small DNA content changes are to be detected with flow karyotyping.     

Analysis of CYP1A1 exon 7 polymorphisms by PCR-SSCP in a Brazilian population and description of two novel gene variations

CYP1A1 polymorphisms have been associated with a higher risk to develop lung cancer, particularly in Japanese. The type and the frequency of the polymorphisms can vary according to the ethnicity. In the present study, we aimed to determine the frequency of CYP1A1(*)2B and (*)4, and to look for other possible polymorphisms that may happen in exon 7 in individuals from Rio de Janeiro, an ethnic mixed population from Brazil. We developed a PCR-SSCP method for screening the genomic polymorphic region from 2289 to 2645 bp. Seven different migration patterns were found among 405 individuals, 130 healthy blood donors and 275 outpatients from Hospital Universitário Pedro Ernesto located in Rio de Janeiro. Five of the migration patterns corresponded to the genotypes: (*)1/(*)1 (the wild type); (*)1/(*)2B; (*)1/(*)4, (*)2B/(*)4 (heterozygous polymorphic) and (*)2B/(*)2B (homozygous polymorphic). Two other patterns corresponded to gene alterations not yet published: a C > T transition localized at the position 2461, and a C > T transition localized at position 2445. The genotype frequencies of the studied polymorphisms were: for CYP1A1(*)2B - 83.7% to (*)1/(*)1, 15.1% to (*)1/(*)2B and 1.2% to (*)2B/(*)2B; for CYP1A1(*)4 - 93.1% to (*)1/(*)1, 6.9% to (*)1/(*)4. The distribution of CYP1A1(*)2B and (*)4 genotypes combined were similar between white and non-white individuals. However, when the non-white individuals were stratified between blacks and mulattos, and then compared with white, black individuals showed a higher frequency of the wild type genotype (P = 0.008) and a lower frequency of genotype (*)1/(*)4 (P = 0.026). Additionally, when black and mulatto individuals were compared, blacks had a higher frequency of the wild type genotype (P = 0.008) and a lower frequency of the(*)1/(*)2B genotype (P = 0.0008), showing a different ethnic distribution of CYP1A1 polymorphisms.     

Frequency of fokI and taqI polymorphism of vitamin D receptor gene in Indian population and its association with 25-hydroxyvitamin D levels

BACKGROUND:

The VDR protein is at the centre of the vitamin D endocrine system, a complex physiological system with substantial feedback regulatory mechanisms involved in maintaining serum calcium and 1, 25 dihydroxy vitamin D3. Variations in VDR gene are shown to have implications in several diseases and have also been implicated as an important genetic factor affecting bone mass.

AIM:

To determine the frequency of Fok I and Taq I variants in healthy Indian individuals and its association with 25-OH-Vitamin D levels.

SETTINGS AND DESIGN:

Blood samples were collected from 143 unrelated normal individuals (Male-84 and Female-59) and their genotypes determined.

MATERIALS AND METHODS:

After amplification by polymerase chain reaction, each polymorphism was genotyped by restriction fragment length polymorphism. For 100 normal healthy individuals 25-hydroxyvitamin D estimation was done using DiaSorin kit method.

STATISTICAL ANALYSIS:

Graph pad software was used to calculate the P values from the Chi-square.

RESULTS:

Out of 143 samples analyzed for FokI and TaqI polymorphisms the following genotypic frequency was obtained FF 59%, Ff 36%, ff 5% and TT 49%, Tt 43%, tt 8% respectively.

CONCLUSIONS:

Results indicate that the distribution of the polymorphic loci Fok I and Taq I vary considerably not only in different populations, but also within India. Furthermore, when the genotypes were analyzed with respect to 25-OH-Vitamin D levels, a significant association was seen for the Taq 1 SNP but not with the Fok I.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3.

The region including the conserved Ser65-Asp66 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon Tn10 is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T. (1990) J. Biol. Chem. 265, 15525-15530). The dipeptide is in putative interhelix loop2-3, which also includes the conserved sequence motif, GXXXXRXGRR, found in all TET proteins and sugar/H+ symporters. Through the combination of localized random and site-directed mutagenesis, each residue in loop2-3 was replaced. Among 10 residues in putative loop2-3, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Gly62, Asp66, Gly69, and Arg70. The defect in the transport activity of the Gly62 and Gly69 substitution mutants corresponded to the steric hindrance by the substituents as to the putative beta-turn structure of the peptide backbone containing these glycines. Of 3 conserved Arg residues, the replacement of only Arg70 caused complete loss of the activity except for replacement with Lys, indicating the importance of a positive charge at this position, which is similar to the essentiality of a negative charge at Asp66. A "charge-neutralizing" intra-loop salt bridge between Asp66 and Arg70 was not likely because the double mutant in which Asp66 and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity. A triple mutant with only one positive charge at Arg70 in this loop showed about half the wild-type activity, indicating that the polycationic nature of the loop was not critical for the activity. Cys mutants as to the unessential residues in the loop were modifiable with N-ethylmaleimide, except for the Met64----Cys and Arg71----Cys mutants; however, the modification of only the Ser65----Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop2-3.     

Relation between sequence similarity and structural similarity in proteins. Role of important properties of amino acids

In a previous paper we obtained ten (orthogonal) factors, linear combinations of which can express the properties of the 20 naturally occurring amino acids. In this paper, we assume that the most important properties (linear combinations of these ten factors) that determine the three-dimensional structure of a protein are conserved properties, i.e., are those that have been conserved during evolution. Two definitions of a conserved property are presented: (1) a conserved property for an average protein is defined as that linear combination of the ten factors that optimally expresses the similarity of one amino acid to another (hence, little change during evolution), as given by the relatedness odds matrix of Dayhoff et al.; (2) a conserved property for each position in the amino acid sequence (locus) of a specific family of homologous proteins (the cytochromec family or the globin family) is defined as that linear combination of the ten factors that is common among a set of amino acids at a given locus when the sequences are properly aligned. When the specificity at each locus is averaged over all loci, the same features are observed for three expressions of these two definitions, namely the conserved property for an average protein, the average conserved property for the cytochromec family, and the average conserved property for the globin family; we find that bulk and hydrophobicity (information about packing and long-range interactions) are more important than other properties, such as the preference for adopting a specific backbone structure (information about short-range interactions). We also demonstrate that the sequence profile of a conserved property, defined for each locus of a protein family (definition 2), corresponds uniquely to the three-dimensional structure, while the conserved property for an average protein (definition 1) is not useful for the prediction of protein structure. The amino acid sequences of numerous proteins are searched to find those that are similar, in terms of the conserved properties (definition 2), to sequences of the same size from one of the homologous families (cytochromec and globin, respectively) for whose loci the conserved properties were defined. Many similar sequences are found, the number of similarities decreasing with increasing size of the segment. However, the segments must be rather long (15 residues) before the comparisons become meaningful. As an example, one sufficiently large sequence (20 residues) from a protein of known structure (apo-liver alcohol dehydrogenase that is not a member of either family) is found to be similar in the conserved properties to a particular sequence of a member of the family of human hemoglobin chains, and the two sequences have similar structures. This means that, since conserved properties are expected to be structure determinants, we can use the conserved properties to predict an initial protein structure for subsequent energy minimization for a protein for which the conserved properties are similar to those of a family of proteins with a sufficiently large number of homologous amino acid sequences; such a large number of homologous sequences is required to define a conserved property for each locus of the homologous protein family.     

Adaptation in sound localization processing induced by interaural time difference in amplitude envelope at high frequencies

Background

When a second sound follows a long first sound, its location appears to be perceived away from the first one (the localization/lateralization aftereffect). This aftereffect has often been considered to reflect an efficient neural coding of sound locations in the auditory system. To understand determinants of the localization aftereffect, the current study examined whether it is induced by an interaural temporal difference (ITD) in the amplitude envelope of high frequency transposed tones (over 2 kHz), which is known to function as a sound localization cue.

Methodology/Principal Findings

In Experiment 1, participants were required to adjust the position of a pointer to the perceived location of test stimuli before and after adaptation. Test and adapter stimuli were amplitude modulated (AM) sounds presented at high frequencies and their positional differences were manipulated solely by the envelope ITD. Results showed that the adapter''s ITD systematically affected the perceived position of test sounds to the directions expected from the localization/lateralization aftereffect when the adapter was presented at ±600 µs ITD; a corresponding significant effect was not observed for a 0 µs ITD adapter. In Experiment 2, the observed adapter effect was confirmed using a forced-choice task. It was also found that adaptation to the AM sounds at high frequencies did not significantly change the perceived position of pure-tone test stimuli in the low frequency region (128 and 256 Hz).

Conclusions/Significance

The findings in the current study indicate that ITD in the envelope at high frequencies induces the localization aftereffect. This suggests that ITD in the high frequency region is involved in adaptive plasticity of auditory localization processing.     

Intronic alternative splicing regulators identified by comparative genomics in nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.     

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo

Background

Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested.

Results

The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1–2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (ΔORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404–459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent.

Conclusions

The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1–2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.     

Tracing disease gene(s) in non-syndromic clefts of orofacial region: HLA haplotypic linkage by analyzing the microsatellite markers: MIB, C1_2_5, C1_4_1, and C1_2_A

BACKGROUND:

Cleft lip with or without cleft palate (CL/P) is the most frequent craniofacial malformation seen in man. The etiology of CL/P is complex involving both genetic and epigenetic (environmental) factors, and the genes play an almost deterministic role in the normal development of craniofacial structures. This study was aimed at ascertaining the association of HLA microsatellites in CL/P patients.

MATERIALS AND METHODS:

Case DNA was obtained from 76 patients (40M and 36 F, average age 7.8 years, range 1-16 years). Unaffected individuals from the same geographical area without population mixing included as controls (n=154, 76 M and 78 F, average age 8.2 years, range 2-17 years). All DNA samples were purified from peripheral blood by standard techniques.

RESULTS:

Four microsatellites were compared in this case-control study. C1_2_5 locus was the most polymorphic marker with 15 observed alleles while C1_4_1 had the least number of alleles. Three of the four markers viz MIB,C1_4_1 and C1_2_5 showed a significant association of microsatellite alleles with CL/P. Five alleles (MIB_326,332,350; C1_4_1 – 213 and C1_2_5-204) were seen with an increased frequency among the test samples, whereas two alleles (C1-4_1_217, and C1_2_5_196) had an increased frequency among the control samples. One allele (C1-4-1-209) had an increased frequency in patient group but was not observed in the controls.

CONCLUSION:

The role of HLA complex in the pathogenesis of CL/P is speculative and has not been established so far. The result of this study shows that a few alleles have an increased frequency of expression in the diseased group which suggests that these alleles may predispose the individuals to clefting. This finding may be beneficial to aid in early diagnosis and plan intervention strategies.     

The effect of conditional probability of chord progression on brain response: an MEG study

Background

Recent electrophysiological and neuroimaging studies have explored how and where musical syntax in Western music is processed in the human brain. An inappropriate chord progression elicits an event-related potential (ERP) component called an early right anterior negativity (ERAN) or simply an early anterior negativity (EAN) in an early stage of processing the musical syntax. Though the possible underlying mechanism of the EAN is assumed to be probabilistic learning, the effect of the probability of chord progressions on the EAN response has not been previously explored explicitly.

Methodology/Principal Findings

In the present study, the empirical conditional probabilities in a Western music corpus were employed as an approximation of the frequencies in previous exposure of participants. Three types of chord progression were presented to musicians and non-musicians in order to examine the correlation between the probability of chord progression and the neuromagnetic response using magnetoencephalography (MEG). Chord progressions were found to elicit early responses in a negatively correlating fashion with the conditional probability. Observed EANm (as a magnetic counterpart of the EAN component) responses were consistent with the previously reported EAN responses in terms of latency and location. The effect of conditional probability interacted with the effect of musical training. In addition, the neural response also correlated with the behavioral measures in the non-musicians.

Conclusions/Significance

Our study is the first to reveal the correlation between the probability of chord progression and the corresponding neuromagnetic response. The current results suggest that the physiological response is a reflection of the probabilistic representations of the musical syntax. Moreover, the results indicate that the probabilistic representation is related to the musical training as well as the sensitivity of an individual.     

Conservation of human immunodeficiency virus type 1 gp120 inner-domain sequences in lentivirus and type A and B retrovirus envelope surface glycoproteins

We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47–54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand β25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for β-strand 5 in the C2 region, which is antiparallel to β25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for β5 and part of the preceding strand β4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in β4. In addition, the β4/β5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel β-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.Lentiviruses are a group of strictly exogenous retroviruses that infect a range of mammalian hosts. One characteristic of this group of retroviruses is the rapid sequence divergence observed between virus strains as well as different lentiviruses, which resulted in the evolution of viruses with large differences in genome organization and sequence (20). Most of the sequence homology between highly divergent lentiviruses is present in the gag and pol gene products (8, 21). Sequence homology between the envelope glycoproteins of different lentiviruses has previously been shown to occur only in the ectodomain of the transmembrane subunit (TM) but not in the surface unit (SU) glycoprotein (3, 8, 21–23). Due to this apparent lack of sequence conservation in lentiviral SU, it has been unclear how the SU of different lentiviruses are structurally related to each other. To address this question, we recently compared SU sequences from the gp120 from primate lentiviruses and the gp135 of small ruminant lentiviruses and found a statistically significant sequence similarity between the second conserved region (C2) of gp120 and a 99-amino-acid region from gp135 (10). Analysis of this gp120-gp135 sequence similarity in the context of the gp120 structure revealed a partial structural similarity between gp120 and gp135.The human immunodeficiency virus type 1 (HIV-1) gp120 core bound to CD4 is composed of two major domains, the inner and outer domains, and a minidomain composed of four antiparallel β-strands, the bridging sheet (13). Sequences from the C2 region form most of the β-strands of a two-helix, two-strand bundle and a five-stranded β-sandwich in the inner domain as well as some β-strands of the outer domain of gp120 (13). Most of the similarity motifs between gp135 and the C2 region of gp120 coincide with sequences corresponding to β-strands 4 through 8 in the HIV-1 gp120 inner domain and β-strands 11 and 12 in the outer domain (10). Significantly, all four cysteines that form two disulfide bonds in the proximal region of the gp120 inner domain as well as the first cysteine of the gp120 V3 loop in β12 (13, 15) are conserved in gp135, indicating a partial similarity between the tertiary structures of gp120 and gp135 (10).The most conserved sequences between gp120 and gp135 correspond to strands β4 and β5 in the five-stranded β-sandwich structure of the proximal region of the inner proximal domain of HIV-1 gp120. Two additional β-strands in this five-stranded β-sandwich are derived from C1 and C5 sequences of HIV-1 gp120 (13). We hypothesized that C1 and C5 sequences, which are part of a structurally conserved SU inner proximal domain, should also be conserved between gp120 and gp135 and possibly in the SU of other lentiviruses. Here we show that two short motifs located in the gp120 C2 and C5 regions which are part of an antiparallel β-sheet in the gp120 inner proximal domain are conserved in the lentiviruses, indicating that a surface of the inner domain of HIV-1 gp120 is conserved in the SU of other lentiviruses. In addition, the C2 motif is also present in the envelope glycoproteins encoded by A-type endogenous retroviral elements and type B retroviruses (type A and B retroviruses), suggesting a local structural similarity between the SU of lentiviruses and type A and B retroviruses.

Sequence motif of the C5 region of HIV-1 gp120 is present in the SU of all lentiviruses.

As the sequences of three of the five β-strands of the gp120 inner proximal domain β-sandwich are conserved in gp135, we first tried to determine whether the gp120-gp135 sequence similarity extends to the other two β-strands which are part of this structure. One of these strands is β1, located in the C1 region of gp120 (13). Although the sequence of β1 is relatively well conserved among the primate lentiviruses, it is only 3 amino acids long, and a reliable assignation of similar sequences in gp135 could not be done. The other strand of this β-sandwich structure is the 7-amino-acid long β25. This strand is antiparallel to β5, which is the most conserved sequence between gp120 and gp135 (10, 13). Strand β25 is located about 20 amino acid residues upstream from the carboxy terminus of HIV-1 gp120 in the C5 region, and its sequence is highly conserved among strains of primate lentiviruses (sequence KYKVVKI in HIV-1_HXB2; residues conserved between HIV-1 strains are underlined) (12, 24). The last residue of this motif has been shown to be important for anchoring of gp120 on gp41 (9), suggesting that β25 is a functionally important structure of the inner proximal domain of gp120 likely to be conserved in other lentiviral glycoproteins. Sequences similar to the HIV-1 gp120 β25 motif (C5 motif) were visually sought in the gp135 carboxy-terminal region. A similar sequence was found in the caprine arthritis-encephalitis virus (CAEV) and visna virus gp135 between 33 and 34 amino acid residues upstream from the carboxy terminus of gp135 (Fig. ​(Fig.1B).1B). Similar to the C5 motif sequence of primate lentiviruses, the gp135 C5 motif is highly conserved in the gp135 of small ruminant lentiviruses (4, 27, 31, 35, 36). The sequence similarity also included the strictly conserved residue L483 of HIV-1 gp120 in the preceding α-helix 5, which is part of the two-helix, two-strand bundle of the inner domain. Flanking regions of gp120 and gp135 did not show any sequence similarity (not shown). Due to its short length, the significance of the conservation of the C5 motif in gp120 and gp135 was unclear. If this motif is indeed part of a structurally or functionally important domain of SU and not due only to chance, it should also be conserved in the SU of other lentiviruses. Therefore, to establish the relevance of this sequence similarity, we determined whether the C5 motif was also present in the carboxy terminus of the SU of other lentiviruses. Open in a separate windowFIG. 1Alignment of the C2 (A) and C5 (B) motifs of the SU from lentiviruses and type A and B retroviruses. Numbers at the right of the alignments indicate the position of the last residue of the motif from the initiation codon. Letters above the alignment indicate residue positions within each motif. Black backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses for each position of the motifs. Gray backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses (but which are nonconservative with the residues in black background) for each position. Numbers in parentheses indicate the number of amino acids between the last position of the C5 motif and the carboxy terminus of SU for each lentivirus. Thick lines indicate sequences which are part of HIV-1 gp120 strands β4, β5, and β25 and helix α5 (13). HIV-1 and HIV-2, human immunodeficiency virus types 1 (strain HXB2, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"K03455","term_id":"1906382"}}K03455) and 2 (strain ROD, {"type":"entrez-nucleotide","attrs":{"text":"X05291","term_id":"60146"}}X05291); CAEV, caprine arthritis-encephalitis virus ({"type":"entrez-nucleotide","attrs":{"text":"M33677","term_id":"323294"}}M33677); Visna, visna virus ({"type":"entrez-nucleotide","attrs":{"text":"M10608","term_id":"470313"}}M10608); JSRV, jaagsiekte sheep retrovirus ({"type":"entrez-nucleotide","attrs":{"text":"M80216","term_id":"331338"}}M80216); EIAV, equine infectious anemia virus (AF033820); FIV, feline immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M73965","term_id":"323946"}}M73965); BIV, bovine immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M32690","term_id":"210706"}}M32690); JDV, jembrana disease lentivirus ({"type":"entrez-nucleotide","attrs":{"text":"U21603","term_id":"733067"}}U21603); HERV-K, human endogenous retrovirus K, type 2 genome ({"type":"entrez-nucleotide","attrs":{"text":"X82272","term_id":"757871"}}X82272); MMTV, mouse mammary tumor virus ({"type":"entrez-nucleotide","attrs":{"text":"X01811","term_id":"61630"}}X01811); MIAE, mouse intracisternal A-type element ({"type":"entrez-nucleotide","attrs":{"text":"M73818","term_id":"194061"}}M73818).Sequences conforming to the C5 motif consensus were also found in the SU of the equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and the bovine jembrana disease lentivirus (JDV), 19 to 23 amino acid residues from the carboxy terminus of SU, the same relative position as the C5 motif from the carboxy terminus of gp120 in primate lentiviruses (Fig. ​(Fig.1B).1B). This sequence similarity was clear when considering the chemical similarities of amino acid side chains (Gln/Glu, Tyr/Trp, Lys/Arg/Gln, Val/Leu, or Val/Ile). A survey of lentiviral SU sequences present in GenBank revealed that the C5 motif was also highly conserved between EIAV, FIV, and BIV strains. For example, the C5 motif was found to be strictly conserved in 64 of 69 EIAV gp90 sequences in GenBank and is also stable during in vivo persistent infection (16, 39). However, the little variation that is observed between strains of a given lentivirus follows the same pattern as variation between different lentiviruses, suggesting a common constraint on sequence variation in different lentiviruses. For example, position h of the C5 motif of HIV-1 gp120 can be either of the conservative variations Lys/Arg or Gln/Glu, the same amino acids present at position h in other lentiviruses. Similarly, position h of the C5 motif of CAEV in different strains is either Lys or Arg (35), two of the residues allowed at position h in HIV-1 gp120. In addition, position b in the C5 motif of most FIV gp100 sequences in GenBank is the conservative variation Gln or Glu, the same amino acids present at position b of the C5 motif in EIAV and the small ruminant lentiviruses, respectively. Although the C5 motif is present in all lentiviruses, the flanking sequences were not consistently conserved except for a few amino acids in some pairwise alignments (not shown). Therefore, although conservation of the C5 motif may not be statistically significant in some SU pairwise alignments, the presence of this motif in the same position relative to the carboxy terminus of SU in all lentiviruses indicates that strand β25 of gp120 is an important structural or functional domain conserved in all lentiviruses.

Sequences similar to an HIV-1 gp120 C2 motif are present in the SU of most lentiviruses.

Using computer-assisted searches, we were previously unable to find in EIAV, BIV, or FIV the same extensive region of similarity that is observed between the C2 region of gp120 and gp135 (10). However, the presence of the C5 (β25) motif in all lentiviruses suggests that sequences similar to gp120 β5, which is antiparallel to β25 and conserved between gp120 and gp135, are also present in degenerate form in other lentiviruses. Visual examination of SU sequences from different lentiviruses revealed the presence of a similar motif (C2 motif) in EIAV, BIV, and JDV although not in FIV (Fig. ​(Fig.1A).1A). This 12-amino-acid C2 motif encompasses most of gp120 β-strands 4 and 5 and includes a strictly conserved cysteine residue in the β4 region. The C2 motif is highly conserved between strains of EIAV and BIV. In EIAV, the C2 motif is stable during persistent infection, with few conservative changes observed (16, 39). In addition, the C2 motif was found to be strictly conserved in 176 of 179 EIAV gp90 sequences present in GenBank, despite considerable sequence variation in other regions.Although some positions of the C2 motif were not absolutely conserved, we found a common pattern of variation between distantly related lentiviruses. For example, position f of the C2 motif is either Pro in EIAV, HIV-2, and simian immunodeficiency virus or aromatic (Tyr or Trp) in the small ruminant lentiviruses BIV and JDV. Also, position h can be either Phe or Tyr even in closely related lentiviruses (HIV-1/HIV-2, visna virus/CAEV, or BIV/JDV), and position l can be either Arg or Lys in the primate and small ruminant lentiviruses or Gln, which is a common conservative substitution for Arg and Lys, in EIAV, BIV, and JDV. Therefore, the C2 motifs of different lentiviruses appear to have a common constraint on sequence variation, suggesting a structural or functional similarity between the HIV-1 gp120 C2 domain and the SU of EIAV, BIV, and JDV.The other previously described gp120-gp135 conserved motifs outside the β4/β5 region could not be identified in the SU of other lentiviruses, including the sequence of gp120 β8, which has a cysteine forming a disulfide bond with the conserved β4 cysteine. Although the C2 motif was not present in FIV gp100, a similar motif was identified in a location upstream from the FIV gp100 V3 region (sequence SYCTDPLQIPLI, amino acids 318 to 329; conserved residues are underlined), in a similar relative position from gp100 V3 as the C2 motif from the V3 region of HIV-1 gp120. However, some of the highly conserved positions of the motif (positions g, h, and j) were not conserved in FIV gp100, and the significance of this FIV gp100 motif is unclear.

C2 motif is present in type A and B retroviral envelope surface glycoproteins.

The conservation of two short motifs in distant regions of SU that are located close to each other in the tertiary structure of HIV-1 gp120 suggests that this region represents a domain of SU that is of structural or functional importance. The TM ectodomains from lentiviruses and type B retroviruses have been shown to have some sequence similarity (19, 34, 38). Therefore, we asked whether sequence similarity between the Env of lentiviruses and type B retroviruses extends to the C2 and C5 motifs of SU.The type A and B retroviruses have some sequence homology in SU, and most of the sequence homology is located in the carboxy-terminal region of SU (18, 38). Visual examination of SU sequences from the human endogenous retrovirus K (18), mouse intracisternal A-type element (26), the exogenous/endogenous mouse mammary tumor virus (25), and the exogenous/endogenous type B/D jaagsiekte sheep retrovirus (JSRV) and the closely related ovine enzootic nasal tumor virus (which encode type B retroviral envelopes) (6, 38) revealed a sequence closely related to the C2 motif in their conserved carboxy-terminal region (Fig. ​(Fig.1A).1A). This sequence represents one of the most conserved sequences in the SU of this group of retroviruses and is also conserved among different strains or members of endogenous families (not shown). Some positions of the C2 motif, such as positions c, d, and g, are strictly or almost completely conserved between the lentiviruses and type A and B retroviruses. However, more informative than the sequence similarity between lentiviruses and type A and B retroviruses is the lack of distinction between the patterns of sequence variation for each position of the motif within and between retrovirus groups, even between closely related viruses. For example, position e of the C2 motif within both the lentiviruses and type A and B retroviruses can be either Pro or basic/Gln; the “dimorphic” position f encodes only Tyr/Trp or Pro (except in HIV-1); position h encodes either Phe or Tyr in all sequences; position i encodes either Ala or a hydrophobic residue in most sequences; position j encodes either Ile, Leu, or Phe in all sequences; position k encodes either Leu, Ile, or Val in all sequences; and position l is preferentially Lys, Arg, or Gln in the lentiviruses and JSRV. Most of these degenerate positions represent very conservative variations (positions a and h through l) or a restricted number of nonconservative variations (positions e and f, in the turn between β4 and β5). The sequence conservation and common pattern of variation between the C2 motifs of lentiviruses and type A and B retroviruses indicate a similar structural or functional constraint on sequence variation in the SU of these two groups of viruses.In contrast to the type A and B retroviruses, sequences similar to the C2 or C5 motifs could not be found in the SU of the Moloney murine leukemia virus, bovine leukemia virus, human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), Rous sarcoma virus, feline RD114 endogenous retrovirus, baboon endogenous retrovirus, feline leukemia virus type A, the Mason-Pfizer monkey retrovirus, or any spumaretrovirus even when using the Findpatterns program of the GCG package (7).Here we show that two short SU motifs are highly conserved in the lentiviruses and that one of these motifs is also conserved in the type A and B retroviruses. Many of the pairwise alignments were not statistically significant when tested by the Monte Carlo simulation of the Bestfit program of the GCG package and could therefore be attributed to chance. However, when all lentiviral sequences are included in the analysis and the multiple alignment is interpreted in the context of the X-ray structure of HIV-1 gp120, the conserved C2 and C5 motifs have a clear structural significance. The conservation of these motifs indicates that the region of the HIV-1 gp120 inner proximal domain centered on the antiparallel β-strands 5 and 25 forms a highly conserved lentiviral SU surface and suggests a possible structural similarity between the SU of lentiviruses and type A and B retroviruses in that domain. Although the C2 motif is too short to rule out convergent evolution between the SU of lentiviruses and type A and B retroviruses, their sequence similarity in TM (19, 34, 38) supports a common origin for most or the entire env genes of these two retroviral groups.The reason for the disagreement between the different degrees of sequence similarity in the SU of lentiviruses and the phylogenetic analyses of the pol gene products is unclear but probably reflects differences in evolutionary rates in different lentiviruses or recombination events (19, 20). Precedents for recombination events between env genes of closely or distantly related retroviruses, deduced from phylogenetic analyses, have been described. An exchange of env sequences probably occurred between HTLV-1 and HTLV-2 (19) and between a type C retrovirus closely related to the avian reticuloendotheliosis virus and a type B retrovirus which originated the type D retroviruses (19, 38).Modeling of the trimeric SU complex on the virion surface indicates that strands β5 and β25 form part of the most virion-proximal surface of the gp120 core (14, 37). While none of the residues of the C2 motif was directly tested for interactions with TM, at least one of the residues of β25 in the C5 region of HIV-1 gp120, I491, is important for stable SU-TM interactions (9). Therefore, the conserved lentiviral SU surface may represent a common structure among lentiviruses and possibly type A and B retroviruses for anchoring SU on TM in the envelope glycoprotein complex. It is interesting that the C5 motif region, which forms a β-strand in the CD4-bound gp120 core, is included in a computer-modeled pocket structure postulated to be important in SU-TM interactions (28), suggesting a structural basis for SU shedding upon receptor-induced conformational change.The sequence of the HIV-1 gp120 outer domain, shown as a cross-hatched box in Fig. ​Fig.2,2, is included entirely between the C2 and C5 motifs (13). Our previous sequence analysis indicates that the gp135s of small ruminant lentiviruses have a similar inner/outer domain organization: most strands of the inner domain β-sandwich as well as β12, located in the outer domain immediately upstream from the gp120 V3 loop, are conserved between gp135 and the gp120 of primate lentiviruses (10). The identification of a homologue of gp120 β25 in gp135 about 290 amino acid residues downstream from the C2 motif provides further support for a similar domain organization in the SU of primate and small ruminant lentiviruses. Consistent with this interpretation, the putative outer domain of gp135, located between the C2 and C5 motifs, is highly glycosylated and contains more than 80% of the potential N-linked glycosylation sites of gp135 (11), similar to the heavy glycosylation of the gp120 outer domain (37). In this gp135 domain model, the distance between the C2 and C5 motifs in the primary structure of SU would indicate a larger relative size of the putative outer domain of gp135 than gp120 outer domain. The presence of the C2 and C5 motifs in EIAV, BIV, and JDV would also suggest an analogous inner/outer domain organization for the SU of these lentiviruses. However, the shorter sequence between the C2 and C5 motifs in EIAV, BIV, and JDV may indicate either a much smaller or absent outer domain in the SU of these viruses (Fig. ​(Fig.2).2). The conserved C2 motif of EIAV gp90 was shown to be part of a minor neutralization epitope recognized by a murine monoclonal antibody (1), suggesting that the EIAV C2 motif is better exposed on the virion surface than the C2 motif of gp120, compatible with a smaller or absent outer domain in gp90. Interestingly, the C2 motif of type A and B retroviruses is located in the carboxy terminus of SU (Fig. ​(Fig.2)2) and C5 appears to be absent, indicating that the surface glycoproteins of type A and B retroviruses, although possibly structurally related to the SU of lentiviruses, probably lack an outer domain homologue and have a different domain organization than the SU of lentiviruses. Open in a separate windowFIG. 2Location of the C2 and C5 motifs in retrovirus envelope glycoproteins. The Env glycoproteins (excluding the amino-terminal leader peptide) are drawn to scale and aligned by the SU-TM cleavage sites conserved in all retroviruses (dotted line). The SU and TM domains of Env are indicated by double arrows. The boundaries of the C2, V3, and C5 regions of HIV-1 gp120 are indicated by thick lines above the alignment, and the location of the HIV-1 gp120 outer domain sequence is shown by a cross-hatched box. The black and gray boxes in the SU domain indicate the positions of the C2 and C5 motifs, respectively. Asterisks represent the described PNDs of EIAV (1), visna virus (29), FIV (17), and T-cell-adapted strains of HIV-1 (33) and HIV-2 (2).The two conserved colinear motifs of lentivirus SU could be useful as structural points of reference for comparative structural studies of SU from different lentiviruses. Variable domains of SU are important in the mechanisms of host cell invasion, tropism determination, and immune evasion. In HIV-1, the third variable loop V3 of gp120 is the main target of neutralizing antibodies in tissue culture-adapted strains and also determines coreceptor usage and tropism (5, 30, 32, 33). Whether sequences in variable regions of SU in other lentiviruses that are functionally equivalent to the gp120 V3 loop are also structurally related to the gp120 V3 loop is not clear. The position of variable domains relative to the C2 and C5 motifs could therefore indicate their structural relationship. For example, the principal neutralization domain (PND) of EIAV gp90, postulated to be functionally equivalent to the gp120 V3 loop (1, 16), is located upstream from the C2 motif instead of downstream, as the V3 loop in gp120 is (Fig. ​(Fig.2),2), suggesting that the gp90 PND and the gp120 V3 loop, while having similar roles in evasion of humoral immune responses, may not be structurally related to each other. A similar situation also occurs in visna virus, whose PND, located in the carboxy-terminal region of gp135 (29), was previously shown to be structurally unrelated to the HIV-1 gp120 V3 loop (10). This would indicate that different lentiviruses may have evolved different regions of a primordial lentivirus surface glycoprotein to perform similar functions important in virus-host interactions.     

杂色山雀亲代喂食与子代乞食间的行为

在育雏期,晚成鸟的子代一般都是由双亲共同来抚育,子代为了更好地存活,会用自己的方式竞争获得更多的食物和更好的生存空间,同时亲代也会根据子代的乞食信号来分配食物。2011年3～7月采用针孔摄像技术录制了杂色山雀(Parus varius)育雏期巢内亲代与子代间的行为,统计了亲鸟站位、雏鸟站位、雏鸟乞食强度及亲鸟的喂食情况等数据。分析结果表明:(1)雌雄亲鸟在巢中的站位各有特点,雄鸟在整个育雏期都喜欢站在距离巢口较近的位置;雌鸟站位不太固定,前期离巢口相对较远,中期和后期离巢口相对较近;(2)雏鸟离亲鸟越近,乞食强度越大,获得食物的机会就越多;离亲鸟越远的雏鸟越不爱乞食,所以站位对雏鸟的食物获得影响最大;(3)雌鸟承担主要的育雏任务,喂食频率远大于雄鸟;(4)育雏期的不同阶段雏鸟乞食强度、亲鸟喂食频率变化很大:中期雏鸟乞食强度最大,亲鸟喂食频率最高,后期雏鸟乞食强度最弱;(5)整个育雏期雌性亲本没有表现出明显的偏爱行为,但雄性亲本在中、后期更偏爱体型大的雏鸟。可见杂色山雀子代的行为和体型大小影响着亲代的食物分配,亲代也会根据雏鸟日龄调整站位和喂食行为。     

Haptoglobin polymorphism among the tribal groups of southern Gujarat

BACKGROUND:

Gujarat is located at the western most point of the Indian subcontinent. Valsad and Surat districts are part of the ‘tribal belt’of Gujarat and constitute 29.1% of total tribal population of Gujarat. These tribal populations are a rich source of gaining insights in the patterns of genetic diversity and genetico-environmental disorders against the back drop of their ecological, historical and ethnographic aspects.

AIM:

The objectives were to find out a) the genetic diversity among the tribes of Gujarat with reference to haptoglobin (Hp) locus b) the relationship between Hp polymorphism and sickle cell anemia/trait.

MATERIALS AND METHODS:

431 individuals belonging to eight tribal groups were studied for Hp polymorphism using polyacrylamide disc gel electrophoresis (PAGE). Hb*S was screened by dithionate tube turbididy (DTT) test and confirmed using cellulose acetate membrane electrophoresis (CAME).

STATISTICAL ANALYSIS:

Allele frequency was calculated by direct gene counting method. Average heterozygosity and gene diversity were computed using software DISPAN. Analysis of molecular variance (AMOVA) was estimated using software ARLEQUIN version 3.1.

RESULTS AND CONCLUSIONS:

Pattern of allele frequency distribution showed preponderance of Hp² allele in all the eight tribal groups, which is in accordance with its frequency in different populations of Indian subcontinent. Total average heterozygosity (H_T) was found to be low (0.160) but the level of genetic differentiation (G_ST) was found to be moderately high (5.6%). AMOVA analysis indicated least among group variance between west and south Indian populations (-0.04%) indicating the affinities of the tribes of Gujarat with that of Dravidian speaking groups. Analysis of Hp phenotypes among sickle cell anemia/ trait individuals revealed a high frequency of Hp 0-0 phenotype (92.7%) among SS individuals as opposed to only 9.7% among AS individuals, reaffirming the selective advantage of HbAS state in relation to hemolytic disorders.     

Structural and Functional Perturbation of Giardia lamblia Triosephosphate Isomerase by Modification of a Non-Catalytic,Non-Conserved Region

Background

We have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.

Results

In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.

Conclusions

The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.     

Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria

Unlike the and polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the and polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not form a subunit complex by in vitro reconstitution with bacteriochlorophyll. Guided by prior experiments with the LH1 polypeptides of Rb. sphaeroides and Rhodospirillum rubrum, which defined a set of interactions required to stabilize the subunit complex, a series of mutations to the Rb. sphaeroides LH2 polypeptide was prepared and studied to determine the minimal changes necessary to enable it to form a subunit-type complex. Three mutants were prepared: Arg at position –10 was changed to Asn (numbering is from the conserved His residue which is known to be coordinated to bacteriochlorophyll); Arg at position –10 and Thr at position +7 were changed to Asn and Arg, respectively; and Arg at position –10 was changed to Trp and the C-terminus from +4 to +10 was replaced with the amino acids found at the corresponding positions in the LH1 polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Thus, the importance of the C-terminal region, which encompasses conserved residues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for oligomerization of subunits to form LH1 complexes. In one of these mutants, Trp at position –10 was changed to Arg, as found in LH2 at this position, and in the other His at position –18 was changed to Val. The results from these mutants allow us to conclude that the residue at the –10 position is unimportant in subunit formation or oligomerization, while the strictly conserved His at –18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.