Functional metabolic characteristics of sodium hyaluronate in mammal organism

In the review data concerning functional metabolic characteristics of sodium hyaluronate in mammal organism, as well as autologous results concerning the study of its intraarticular action on metabolism of conjunctive tissue in the conditions of experimental arthritis in rabbits are given.     

Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.     

Functional and systematic implications of the ear in golden moles (Chrysochloridae)

Golden moles (Chrysochloridae) are fossorial mammals known to have unusual mallei. The aim of this study was to describe and quantify aspects of the auditory morphology of golden moles in order to determine their systematic and functional implications. Observations were made on skeletal material as well as histological sections. The results of this study do not support the separation of the genus Calcochloris from Amblysomus . It was found that the morphology shared by all the studied genera is indicative of specialization for hearing low frequency sound. The tympanic membrane to stapes footplate ratios, ossicular lever arm ratios and incudomallear joint morphology suggest low frequency specializations in genera with small mallear heads and high frequency specializations in genera with large mallear heads. However, the size and degree of trabeculation of the tympanic cavity are not consistent with this result. It is proposed that all golden moles are low frequency hearers with differences in their range of sensitivity according to how much time they spend foraging above ground.     

Functional analysis of an endo-1,6-beta-D-glucanase gene (neg-1) from Neurospora crassa

The 1,6-beta-D-glucanase gene (neg1) of Neurospora crassa was disrupted by repeat-induced point mutations. Sequence analysis of the neg1 gene in the R12-1 mutant showed that 9 nucleotides within the coding region of the gene changed from GC to AT. The base transition of C to A at position 662 resulted in a codon. No apparent phenotypic changes were observed in the mutant, but Congo-red, SDS, and cetyltrimethyl ammonium bromide (CTAB), which affect fungal cell walls or membranes, markedly inhibited the hyphal growth of the mutant at a concentration that does not inhibit growth in the wild type.     

Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.     

Functional analysis of the Streptomyces lividans type I signal peptidases

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.     

Functional analysis of the triplex proteins (VP19C and VP23) of herpes simplex virus type 1

The triplex of herpesvirus capsids is a unique structural element. In herpes simplex virus type 1 (HSV-1), one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent VP5 molecules. The interaction between VP19C and VP23 was inferred by yeast cryoelectron microscopy studies and subsequently confirmed by the two-hybrid assay. In order to define the functional domains of VP19C and VP23, a Tn7-based transposon was used to randomly insert 15 bp into the coding regions of these two proteins. The mutants were initially screened for interaction in the yeast two-hybrid assay to identify the domains important for triplex formation. Using genetic complementation assays in HSV-1-infected cells, the domains of each protein required for virus replication were similarly uncovered. The same mutations that abolish interaction between these two proteins in the yeast two-hybrid assay similarly failed to complement the growth of the VP23- and VP19C-null mutant viruses in the genetic complementation assay. Some of these mutants were transferred into recombinant baculoviruses to analyze the effect of the mutations on herpesvirus capsid assembly in insect cells. The mutations that abolished the interaction in the yeast two-hybrid assay also abolished capsid assembly in insect cells. The outcome of these experiments showed that insertions in at least four regions and especially the amino terminus of VP23 abolished function, whereas the amino terminus of VP19C can tolerate transposon insertions. A novel finding of these studies was the ability to assemble herpesvirus capsids in insect cells using VP5 and VP19C that contained a histidine handle at their amino terminus.     

Functional analysis of the TFIID-specific yeast TAF4 (yTAF(II)48) reveals an unexpected organization of its histone-fold domain

Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAF(II)s), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAF(II)48) containing the HFD shares strong sequence similarity with Drosophila (d)TAF4 (dTAF(II)110) and human TAF4 (hTAF(II)135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperature-sensitive mutations in the yTAF4 HFD alpha2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAF(II)68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the alpha3 region within the predicted TAF4 HFD is unstructured and does not correspond to the bona fide alpha3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD alpha3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the alpha3 helix is separated from the alpha2 helix by an extended loop containing a conserved functional domain.     

Functional cranial analysis of large animalivorous bats (Microchiroptera)

Large animalivorous bats include carnivorous, piscivorous and insectivorous microchiropterans. Skull proportions and tooth morphology are examined and interpreted functionally. Four wide- faced bats from four families are convergent in having wide skulls, large masseter muscle volumes and stout jaws, indicating a powerful bite. Three of the four also have long canine teeth relative to their maxillary toothrows. Carnivorous bats have more elongate skulls, larger brain volumes and larger pinnae. The wide-faced bats are all dral emitters and have heads positively tilted relative to the basicranial axis. The carnivorous species are nasal-emitting bats and have negatively tilted heads. The orientation of the head relative to the basicranial axis affects several characters of the skull and jaws and is not correlated with size. The speculation that the type of echolocation may be more of a determinant of evolutionary change than the feeding mechanism is addressed. Wide-faced bats are thought to be capable of eating hard prey items (durophagus) and are probably non- discriminating, aurally less sophisticated insect generalists while the carnivorous and non- durophagus insectivorous bats may be more discriminating and aurally more sophisticated in what they eat.     

Functional analysis of chimpanzee (Pan troglodytes) private signing

The chimpanzee's use of American Sign Language (ASL) to communicate with humans and with each other has been empirically demonstrated in several reports, but this is the first research to experimentally examine their use of sign language in a nonsocial fashion: private signing. This experiment examined the private signing behavior of five signing chimpanzees, using a remote videotaping technique with no human present. It was found that all five chimpanzees signed to themselves for a total of 368 instances. These instances of private signing were classified into nine different functional categories as has been done in the analysis of private speech and signing in hearing and deaf human children. Similar to humans, a few of the categories accounted for the majority of the instances of private signing. These findings empirically demonstrate a behavior similar to private speech and signing in humans.     

Genetic analysis of aMicroseris douglasii (Asteraceae) population polymorphic for an alien chloroplast type

Recent evidence suggests chloroplast introgression fromMicroseris bigelovii intoM. douglasii. We have examined 23 plants from a population ofM. douglasii polymorphic forM. douglasii andM. bigelovii chloroplast types. All 23 plants were completely homozygous for morphological and RAPD markers, and inbred lines derived by selfing have been used for DNA analysis. Chloroplast RFLP analysis identified 16 plants withM. bigelovii chloroplasts and seven withM. douglasii chloroplasts. The nuclear genomes of the 16 plants withM. bigelovii chloroplasts were examined with 22 primers for RAPD amplification products shared exclusively withM. bigelovii. Five of 268 markers appeared to be shared betweenM. bigelovii and one or more of these 16 plants on the basis of their position in gels. Detailed examination of these five amplification products showed that none of them are nuclear DNA fromM. bigelovii. Very little, if any, nuclear DNA fromM. bigelovii can be present inM. douglasii plants with chloroplasts typical ofM. bigelovii. The study demonstrates the usefulness of the RAPD technique for screening large numbers of markers to select a few potentially informative ones for rigorous examination.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Induction of diploid gynogenesis in an evolutionary model organism, the three-spined stickleback (Gasterosteus aculeatus)

Background

X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities.

Results

We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients.

Conclusions

There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution.     

Functional characteristics of hexokinase bound to the type a and type B sites of bovine brain mitochondria.

Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, approximately 40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of Type A:Type B sites, which varies in different species.