Atlanto-occipital fusion: an osteological study with clinical implications.

BACKGROUND: Atlanto-occipital fusion may be symptomatic or asymptomatic in nature. The anomaly may be incidentally detected at autopsies or during routine cadaveric dissections. The fusion of the atlas with occipital bone may result in the compression of vertebral artery and first cervical nerve. METHODS: A total of 55 dried occipital bones in the Department of Anatomy, Universiti Kebangsaan Malaysia (UKM) and Department of Anatomy, Universiti Malaya (UM) were included in the study. The presence of atlantooccipital fusion was closely observed and morphometric measurements were taken. RESULTS: Out of 55 dried occipital bones studied, we observed atlanto-occiptalization in two bones (3.63 %). A total of 53 occipital bones (96.37 %) did not exhibit any anomalous fusions. Out of the two anomalous atlanto-occiptal fusions, one was complete while the other had unilateral right-sided fusion of the atlas with the occipital bone. CONCLUSION: Atlanto-occipitalization may result in the compression of vertebral artery thereby influencing the blood flow to the brain. Atlanto-occipitalization may also result in compression of the first cervical nerve. The action of the postural muscles on the extensor surface may be affected as a result of this anomaly. The present article discusses the clinical implications of atlanto-occipitalization, which may be beneficial for neurosurgeons, neurologists and radiologists in day-to-day clinical practice (Fig. 3, Ref. 17).

A multicopy DNA sequence from Meconopsis simplicifolia discriminates between the different species of this endangered Himalayan poppy.

Clones harboring multicopy DNA sequences were isolated on the basis of reverse genome hybridization to Meconopsis paniculata (Himalayan yellow poppy) DNA from a Sau3A partial genomic plasmid library of M. simplicifolia (Himalayan blue poppy). Restriction-endonuclease-dependent genetic polymorphism between five species of Meconopsis, M. aculeata, M. paniculata, M. simplicifolia, M. sinuata and M. villosa, belonging to geographically isolated populations, was evident in genomic DNA filter hybridizations when probed with a clone (pIMS10) isolated from this library. Pooled DNA from seedlings originating from plants of individual populations of M. paniculata, M. simplicifolia and M. villosa gave similar band patterns, with respect to a given enzyme, suggesting intra-population genetic homogeneity.

Genetic alterations in brain tumors identified by RAPD analysis.

We report the utility of random amplified polymorphic DNA (RAPD) analysis for identifying subtle genomic alterations in meningiomas and gliomas by comparing the DNA band profile of tumor vis-à-vis its constitutional counterpart. Twenty out of the 29 decanucleotide GC-rich random primers utilized for the RAPD analysis of meningiomas revealed alteration(s) in the tumor genome. In gliomas, changes were detected by 16 of the 18 primers. While all the seven meningioma samples exhibited alterations in tumor DNA, changes were evident in 21 of the 24 glioma cases. These alterations in tumor DNA included the loss of a normal band, appearance of a new band and amplification of a pre-existing band. Many primers detected more than one alterations in a given tumor. Our approach, which covers the range from 0.4 to 2 kb, besides detecting a significant number of changes in a spectrum of brain tumors, complements existing DNA fingerprinting methods, such as microsatellite mapping (less than 0.4 kb) and Southern blotting (over 2 kb), for detecting genetic alterations in tumors.

Characterization of a human alphoid satellite DNA sequence: potential use in assessing genetic diversity in Indian populations.

Sequence analysis of a human repetitive DNA sequence (pTRF5.6) revealed considerable homology (76%) to the alphoid consensus sequence. Genomic blots of StuI-digested human DNA, hybridized to pTRF5.6, generated a ladder of bands with each band corresponding to oligodeoxyribonucleotide of an approx. 170-bp repeat, indicating a tandemly arrayed organization of this repeat element within the genome. Genomic hybridization analyses of unrelated individuals belonging to various geographical regions of India, using this alphoid satellite prove, revealed polymorphic bands ranging between 2 and 9 kb. Along with an individual-specific band pattern, several isomorphic bands below 2 kb were also evident. There was very little genetic variability between populations, suggestive of low polymorphism at the inter-population level. Our result suggest that alphoid satellite sequence probe can be used in assessing the genetic diversity of various ethnic groups/populations belonging to different geographical regions.

Biallelic polymorphism in the intron region of beta-tubulin gene of Cryptosporidium parasites.

Nucleotide sequencing of polymerase chain reaction amplified intron region of the Cryptosporidium parvum beta-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is in agreement with our previous genotyping data based on the thrombospondin-related adhesion protein (TRAP-C2) gene, indicating these genotype characteristics are linked at 2 genetic loci. Characterization of Cryptosporidium muris and Cryptosporidium serpentis has further shown that non-parvum Cryptosporidium parasites have beta-tubulin intron sequences identical to bovine genotype of C. parvum. Thus, results of this study confirm the lineage of 2 genotypes of C. parvum at 2 genetic loci and suggest a need for extensive characterization of various Cryptosporidium spp.

Molecular characterization of a Cryptosporidium isolate from a black bear.

To further validate the observation of the existence of host-adapted strains of Cryptosporidium parvum, we genetically characterized an isolate of Cryptosporidium parasite from a black bear. Sequence analysis of the ribosomal RNA small subunit and the 70-kDa heat shock protein (HSP70) showed that this parasite represents a new genotype of C. parvum and is related to the C. parvum dog genotype. This finding is helpful for clarifying Cryptosporidium taxonomy.

A redescription of Cryptosporidium galli Pavlasek, 1999 (Apicomplexa: Cryptosporidiidae) from birds.

Cryptosporidium galli Pavlasek, 1999, described from the feces of birds, is redescribed with additional molecular and biological data. Oocysts are ellipsoidal, are passed fully sporulated, lack sporocysts, and measure 8.25 x 6.3 microm (range 8.0-8.5 x 6.2-6.4 microm) with a length-width ratio of 1.30 (n = 50). Oocysts are structurally similar to those of Cryptosporidium baileyi described from chickens, but in addition to being considerably larger than oocysts of C. baileyi, these oocysts infect the proventriculus in a variety of birds and not the respiratory tract. Oocysts were successfully transmitted from chickens to chickens, and morphologically similar oocysts also were observed in a variety of exotic and wild birds (Order Passeriformes, Phasianidae, Fringillidae, and Icteridae). Molecular and phylogenetic analyses at the 18S rRNA, HSP70, and actin gene loci demonstrate that this species is genetically distinct from all known species and genotypes of Cryptosporidium and, thus, was named C. galli.

Effect of storage of blood samples on DNA yield, quality and fingerprinting: a forensic approach.

Various storage treatments on human blood samples have been described with respect to DNA yield, quality and fingerprinting. Blood samples were stored at room temperature, 4 degrees C, -20 degrees C and -70 degrees C for different duration varying from 1, 2, 4 and 8 weeks with or without anticoagulant/preservative (EDTA or Heparin). DNA was isolated from these stored samples, quantitated by spectrophotometry and subjected to DNA fingerprinting using a human alphoid satellite DNA sequence (TRF 5.6) and a 33 mer oligonucleotide (O-chi-1) as probes. The polymorphic DNA bands were apparent between 2 to 9 kb size range and the fingerprints were individual-specific. Our results suggest that higher amount of genomic DNA can be recovered from blood samples stored at temperatures 4 degrees C or below in the presence of EDTA or heparin.

Alterations in brain tumor DNA detected by a fingerprinting probe.

We used a novel DNA fingerprinting probe O-chi-1 (ref. 1) to detect differences in the hybridization pattern of brain tumor DNA and paired normal tissue of a given individual. Representatives of meningiomas (two), glioblastoma multeforme (three) and astrocytoma (one) were studied. Alterations, which included amplification as well as the loss of a normal band in tumor DNA, were observed in four of the six tumours. While the increased intensity of a band can be taken to imply increased copy number, the disappearance of bands could either be due to loss of DNA sequence or rearrangement resulting in different sized bands.

Random amplified polymorphic DNA (RAPD) markers reveal genetic homogeneity in the endangered Himalayan species Meconopsis paniculata and M. simplicifolia.

Random amplified polymorphic DNA (RAPD) marker-based analysis was carried out to study the extent of genetic polymorphism between populations of the two endangered Himalayan poppy species, Meconopsis paniculata and M. Simplicifolia. Of the 90 primers tested, 38 revealed marked inter-species genetic polymorphism between individuals of the two species from geographically isolated populations. However, intra-species genetic homogeneity was also evident with respect to a number of primers both within and between populations. A comprehensive analysis incorporating data from RAPDs, DNA fingerprinting and isozyme pattern was carried out and, based on the presence or absence of bands, three matrices of similarity indices were estimated. These matrices were subsequently utilized in cluster analysis. In order to compare the three clusters generated using these three different marker systems, a Mantel matrix-correspondence test was carried out on the basis of comparisons of co-phenetic values. The overall representation of relationships by cluster analysis was similar for all three marker systems and this was substantiated by high correlations among the three analyses revealed by the Mantel matrix-correspondence test. Our results point to very low or absence of, genetic polymorphism in M. paniculata and M. simplicifolia, and are in broad agreement with our previous observations on genetic diversity of Meconopsis species which point to a genetic basis for the possible extinction of this economically important genus.

DNA polymorphism analysis in five endangered species of Meconopsis (Himalayan poppy) using multi-copy sequence-based probes.

An attempted to understand the genetic basis of extinction of Meconopsis (Himalayan poppy), an endangered genus of ornamental value and confined to alpine Himalayas, is described. Multicopy DNA sequences were isolated from a Sau3A partial genomic plasmid library of M. simplicifolia and M. paniculata. Genetic polymorphism between five species of Meconopsis belonging to geographically isolated populations was evident, albeit at a low level, in genomic Southerns when probed with repetitive DNA clones isolated from this library. Intra-population monomorphic band patterns for different populations of a species was evident with respect to a number of enzymes suggesting very low or no genetic variability and/or low mutation rate within a given population of Meconopsis.

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy, Meconopsis simplicifolia (Papaveraceae).

Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10(-6)M kinetin + 10(-5)M α -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10(-5)M 2,4-dichlorophenoxy acetic acid in place of α -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80-85%) as well as on the surface of small clumps of tissue (15-20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.

Evaluation of Cryptosporidium parvum genotyping techniques.

We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.

Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene.

We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. The Cryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes of C. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus Cryptosporidium.

Sequence differences in the diagnostic target region of the oocyst wall protein gene of Cryptosporidium parasites.

Nucleotide sequences of the Cryptosporidium oocyst wall protein (COWP) gene were obtained from various Cryptosporidium spp. (C. wrairi, C. felis, C. meleagridis, C. baileyi, C. andersoni, C. muris, and C. serpentis) and C. parvum genotypes (human, bovine, monkey, marsupial, ferret, mouse, pig, and dog). Significant diversity was observed among species and genotypes in the primer and target regions of a popular diagnostic PCR. These results provide useful information for COWP-based molecular differentiation of Cryptosporidium spp. and genotypes.

Cryptosporidium species and genotypes in HIV-positive patients in Lima, Peru.

Cryptosporidium parasites from a cross-sectional study conducted in two national hospitals in Lima, Peru were genetically characterized to determine the diversity of Cryptosporidium spp. in HIV-positive people. A total of 2,672 patients participated in this study and provided 13,937 specimens. Cryptosporidium oocysts were detected by microscopy in 354 (13.3%) of the patients. Analysis of 951 Cryptosporidium-positive specimens from 300 patients using a small subunit rRNA-based PCR-RFLP tool identified 6 genotypes; Cryptosporidium hominis was the species most frequently detected (67.5%), followed by C. meleagridis (12.6%) and C. parvum (11.3%). Cryptosporidium canis (4.0%), C. felis (3.3%), and Cryptosporidium pig genotype (0.5%) were also found. These findings indicate that C. hominis is the predominant species in Peruvian HIV-positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients.

Differentiating human from animal isolates of Cryptosporidium parvum.

We analyzed 92 Cryptosporidium parvum isolates from humans and animals by a polymerase chain reaction/restriction fragment length polymorphism method based on the thrombospondin-related anonymous protein 2 gene sequence. Used as a molecular marker, this method can differentiate between the two genotypes of C. parvum and elucidate the transmission of infection to humans.

A multilocus genotypic analysis of Cryptosporidium meleagridis.

Cryptosporidium meleagridis is a common cause of cryptosporidiosis in birds. In addition, recent reports have described the parasite as an etiologic agent of cryptosporidiosis in both immunocompetent and immunocompromised humans. Therefore, it is important to genetically characterize isolates of C. meleagridis from different hosts and geographic areas, and to develop molecular tools to differentiate isolates from various hosts or areas. In this study, a total of 11 isolates of Cryptosporidium meleagridis from both human and avian hosts were examined at three genetic loci: the small-subunit rRNA, 60-kDa glycoprotein precursor, and 70-kDa heat shock protein genes. Two genotypes of C. meleagridis were seen at the small-subunit rRNA locus. These differed from each other by the presence or lack of a heterogeneous copy of the gene and an ATT repeat. The 60-kDa glycoprotein precursor gene divided these eleven isolates of C. meleagridis into six genotypes with high sequence diversity between groups. The highest genetic heterogeneity, however, was seen at the 70-kDa heat shock protein locus, and was primarily present at the 3' end of the gene. This heterogeneity separated eight isolates of C. meleagridis into six genotypes. These data could be useful in the development of molecular tools to promote understanding of the transmission of C. meleagridis in humans.