DNA capturing machinery through spore-displayed proteins.

AIMS: The purpose of this study was to develop a general method for the facile development of a new DNA biosensor which utilizes streptavidin-displayed spores as a molecular machinery. METHODS AND RESULTS: Fluorescence spectroscopy was used as a monitoring tool for the streptavidin displayed on the surface of Bacillus thuringiensis spores and as a diagnosis method for DNA detection. As a proof-of-concept, four pathogenic bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumonia were used for the detection of pathogenic species. In addition, a set of mutant variants of Wilson's disease were also used for the detection of single nucleotide polymorphism (SNP) in this system. CONCLUSIONS: This strategy, utilizing streptavidin-displayed spores, is capable of capturing DNA targets for the detection of pathogenic bacteria and for mutation analysis in Wilson's disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach could be useful as a simple platform for developing sensitive spore-based biosensors for any desired DNA targets in diagnostic applications.

Donor site morbidities resulting from conchal cartilage harvesting in rhinoplasty.

OBJECTIVE: Conchal cartilage is frequently used in rhinoplasty, but donor site morbidity data are seldom reported. This study aimed to investigate the complications of conchal cartilage harvesting in rhinoplasty. METHODS: A retrospective chart review of 372 patients who underwent conchal cartilage harvesting for rhinoplasty was conducted. Data regarding patient demographics, types of nasal deformities, graft usage and complications were analysed. RESULTS: A total of 372 patients who underwent conchal cartilage harvesting for rhinoplasty were enrolled. The harvested conchal cartilage tissues were used in a variety of applications: tip graft, dorsal graft, septal reinforcement and correction of nostril asymmetry. Nine cases (2.4 per cent) with donor site morbidities were identified, including four cases (1.1 per cent) with keloids and five cases (1.3 per cent) with haematomas. CONCLUSION: Conchal cartilage harvesting is a safe and useful technique for rhinoplasty, with a low complication rate. However, patients should be informed about the possibility of donor site morbidities such as keloids and haematomas.

Catalysis of NADH-->NADP+ transhydrogenation by adult Hymenolepis diminuta mitochondria.

Hymenolepis diminuta mitochondria catalyze nonenergy-linked and energy-linked NADH-->NADP(+) transhydrogenations, with the latter driven by electron-transport dependent NADH oxidation (electron transport-driven, ETD) or ATP hydrolysis (ATP-driven, ATPD). Using submitochondrial particles, NADH-->NADP(+) transhydrogenations were characterized further. ETD and ATPD reactions were enhanced by bovine serum albumin (BSA) and were inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and niclosamide. The nonenergy-linked reaction was unaffected by these additives. Except for DCCD inhibition of the ATPD reaction, BSA mitigated inhibitor effects on energy-linked activities. BSA enhanced NADH oxidase (but not ATPase) activity. Although DCCD inhibited NADH oxidase and ATPase, BSA only lessened oxidase inhibition. With protonophores, an increase in NADH oxidase (but not ATPase) activity was suggested. Oxidase inhibition by rotenone was unaffected by BSA. The ATP-hydrolyzed/NADPH-formed for the ATPD reaction was almost unity. A model for H. diminuta energy-linked transhydrogenation is presented.

The cloning, genomic structure, localization, and expression of human deoxyribonuclease IIbeta.

Acidic endonuclease activity is present in all cells in the body and much of this can be attributed to the previously cloned and ubiquitously expressed deoxyribonuclease II (DNase II). Database analysis revealed the existence of expressed sequence tags and genomic segments coding for a protein with considerable homology to DNase II. This report describes the cloning of this cDNA, which we term deoxyribonuclease IIbeta (DNase IIbeta) and comparison of its expression to that of the originally cloned DNase II (now termed DNase IIalpha). The cDNA encodes a 357 amino acid protein. This protein exhibits extensive homology to DNase IIalpha including an amino-terminal signal peptide and a conserved active site, and has many of the regions of identity that are conserved in homologs in other mammals as well as C. elegans and Drosophila. The gene encoding DNase IIbeta has identical splice sites to DNase IIalpha. Human DNase IIbeta is highly expressed in the salivary gland, and at low levels in trachea, lung, prostate, lymph node, and testis, whereas DNase IIalpha is ubiquitously expressed in all tissues. The expression pattern of human DNase IIbeta suggests that it may function primarily as a secreted enzyme. Human saliva was found to contain DNase IIalpha, but after immunodepletion, considerable acid-active endonuclease remained which we presume is DNase IIbeta. We have localized the gene for human DNase IIbeta to chromosome 1p22.3 adjacent (and in opposing orientation) to the human uricase pseudogene. Interestingly, murine DNase IIbeta is highly expressed in the liver. Uricase is also highly expressed in mouse but not human liver and this may explain the difference in expression patterns between human and mouse DNase IIbeta.

Hepatosplenic alphabeta T-cell lymphomas: a report of 14 cases and comparison with hepatosplenic gammadelta T-cell lymphomas.

Hepatosplenic gammadelta T-cell lymphoma is a distinct entity, characterized by occurrence in young adult males with hepatosplenomegaly, B-symptoms, peripheral blood cytopenias, and no lymphadenopathy; lymphomatous infiltrates in the splenic red pulp, hepatic sinusoids, and bone marrow sinuses; T-cell receptor (TCR) gammadelta chains and a cytotoxic T-cell phenotype; isochromosome 7q; and an aggressive clinical course. In comparison, this study describes the clinicopathologic features of 14 hepatosplenic T-cell lymphomas expressing TCR alphabeta chains. They occurred in 11 women and 3 men with a median age of 36 years. Clinical presentation was similar to that described previously for hepatosplenic gammadelta T-cell lymphomas, except for the female preponderance and age distribution (5 patients younger than 13 years of age and 5 patients older than 50 years of age). Disease distribution was primarily in the splenic red pulp and hepatic sinusoids, although liver infiltrates were largely periportal in four cases. Bone marrow involvement, observed in eight patients, was usually interstitial and/or within the sinuses. Lymph nodes were involved in five patients, although lymphadenopathy was demonstrable in only two. Ten cases were composed of intermediate-size tumor cells with round/oval nuclei, slightly dispersed chromatin, inconspicuous nucleoli, and scant to moderate amounts of cytoplasm. Four lymphomas contained primarily large cells with irregular nuclei, dispersed chromatin, discernible nucleoli, and moderate to abundant cytoplasm. Tumor cells in all 14 lymphomas were cytotoxic alphabeta T-cells; 13 co-expressed natural killer cell-associated antigens and showed T-cell clonality. Three lymphomas were associated with Epstein-Barr virus. Two of four cases had an isochromosome 7q. Eleven patients are dead, eight within a year of diagnosis, and two patients have maintained complete remissions after combination chemotherapy. These data show that hepatosplenic T-cell lymphomas include an alphabeta-subtype. This group, along with the previously recognized gammadelta group, should be recognized as phenotypically heterogeneous subtypes of the same disease entity.

Mixoploidy in humans: two surviving cases of diploid-tetraploid mixoploidy and comparison with diploid-triploid mixoploidy.

We report on 2 cases of diploid/tetraploid (2n/4n) mixoploidy in surviving females, 11 and 21 years old. Both individuals manifested severe mental retardation, reduced peripheral limb muscle bulk, asymmetric growth deficiency, seizure disorder, and skin pigmentary dysplasia. Previous lymphocyte karyotypes had been normal on 2 occasions, but when skin fibroblast karyotypes were done, 33% of the cells were tetraploid on the younger woman, and over 60% were tetraploid in the older woman (on 2 separate occasions). In both individuals, the distal limbs and digits were long and thin, with reduced small muscle bulk. The similarity in distal limb findings prompted reexamination of the younger woman's chromosomal constitution in skin fibroblasts. We concluded that the clinical findings in these cases are unique and similar, and we caution clinicians about uniformly dismissing tetraploidy as artifactual in amniocytes from normal patients, especially since this phenotype would be very difficult to detect, even with directed prenatal ultrasonography. We compare the 2n/4n phenotype with that in diploid/triploid (2n/3n) mixoploidy and note subtle differences which might be detected postnatally. These findings should be useful in guiding clinicians on when to request skin fibroblast karyotypes in mentally-deficient individuals with asymmetric growth deficiency and pigmentary skin variation.

Ring chromosome 12.

A ring chromosome 12 (p13.3q24.3) was observed in all cells analyzed from skin fibroblasts and the peripheral blood of a 19-year-old man initially referred for developmental delay with expressive language deficiency. Other phenotypic anomalies included growth deficiency, multiple café-au-lait spots, mild pectus excavatum, glandular hypospadias, left esotropia, clinodactyly of the fifth fingers, and hypothyroidism with elevated antithyroid antibodies. The four previously reported cases of r(12) support the theory of a general ring phenotype which is manifested independently of the specific autosome involved and which is characterized by growth failure, moderate mental retardation, and lack of other major phenotypic anomalies. Breakpoints in all cases of r(12) have been assigned to the telomeric regions, suggesting minimal deletion of chromosome material.

Smith-Magenis syndrome resulting from a de novo direct insertion of proximal 17q into 17p11.2.

We report on a de novo intrachromosomal rearrangement of chromosome 17 in a patient with Smith-Magenis syndrome (SMS). This 11-year-old boy had short stature, midfacial hypoplasia, and behavioral problems characteristic of this syndrome. Cytogenetic analysis showed that the proximal long arm of a chromosome 17 (q11.2-q21.3) was inserted into its short arm at p11.2, resulting in an apparent deletion of the SMS critical region [ins(17)(p11.2q11.2q21.3)]. Fluorescence in situ hybridization studies (FISH) demonstrated that the inserted segment included both the ERBB2 and RARA loci, and dual color hybridizations defined the insertion as direct, with ERBB2 located more proximally on the short arm of the der(17). The resulting deletion of the short arm included loci c130G3, D17S258, FLI, and D17S29, while the more proximal loci, D17S446 and D17S58, remained apparently unaffected and in their native locations. The CMT1A locus also remained in its native location on the short arm of the metacentric der(17) chromosome. A de novo intrachromosomal insertional rearrangement of chromosome 17 in a case of SMS has not been reported previously and further illustrates the instability of this chromosomal region.

Familial translocation 5;14 resulting in an unbalanced offspring.

We report on an infant with multiple congenital anomalies possessing a derivative 14 chromosome in excess of the normal complement, resulting from transmission of a familial t(5;14)(p13;q22). The proposita's phenotypically normal mother, mentally retarded half-brother, and fetal sib are carriers of the apparently balanced translocation. Previous cases of similar familial t(5;14) are reviewed. The proposita's phenotype is characterized by failure to thrive, developmental retardation, cleft palate, congenital heart anomaly, abnormal hands and feet, unusual face with abnormal ears, and recurrent respiratory infections. The proposita died at age 9 months and postmortem examination showed multiple central nervous system, cardiopulmonary, gastrointestinal, and genital malformations. Our proposita's phenotype is attributable to contributions from both chromosomes and is consistent with the consequences of both the dup(5p) and dup(14q).

Familial t(11;13)(q21;q14) and the duplication 11q, 13q phenotype.

Cases of duplication of distal 11q or proximal 13q have been reported independently. A specific translocation resulting in duplication of distal 11q, [der(22)t(11;22)(q23;q11)], has been documented in over 40 cases. We report on a male fetus with chromosomal excess of both distal 11q and proximal 13q resulting from a familial translocation. This case supports the causal association of duplication 11q with neural tube defects.

Maternal disomy and Prader-Willi syndrome consistent with gamete complementation in a case of familial translocation (3;15) (p25;q11.2).

Maternal uniparental disomy (UPD) for chromosome 15 is responsible for an estimated 30% of cases of Prader-Willi syndrome (PWS). We report on an unusual case of maternal disomy 15 in PWS that is most consistent with adjacent-1 segregation of a paternal t(3;15)(p25;q11.2) with simultaneous maternal meiotic nondisjunction for chromosome 15. The patient (J.B.), a 17-year-old white male with PWS, was found to have 47 chromosomes with a supernumerary, paternal der(15) consisting of the short arm and the proximal long arm of chromosome 15, and distal chromosome arm 3p. The t(3;15) was present in the balanced state in the patient's father and a sister. Fluorescent in situ hybridization analysis demonstrated that the PWS critical region resided on the derivative chromosome 3 and that there was no deletion of the PWS region on the normal pair of 15s present in J.B. Methylation analysis at exon alpha of the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) gene showed a pattern characteristic of only the maternal chromosome 15 in J.B. Maternal disomy was confirmed by polymerase chain reaction analysis of microsatellite repeats at the gamma-aminobutyric acid receptor beta3 subunit (GABRB3) locus. A niece (B.B.) with 45 chromosomes and the derivative 3 but without the der(15) demonstrated a phenotype consistent with that reported for haploinsufficiency of distal 3 p. Uniparental disomy associated with unbalanced segregation of non-Robertsonian translocations has been reported previously but has not, to our knowledge, been observed in a case of PWS. Furthermore, our findings are best interpreted as true gamete complementation resulting in maternal UPD 15 and PWS.

Paternally derived de novo interstitial duplication of proximal 15q in a patient with developmental delay.

Interstitial duplications of proximal 15q containing the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region have been found in patients with autism or atypical autism. In these cases with an abnormal phenotype, the duplications were maternally derived. Paternal origin of the duplication has been associated with a normal phenotype. We report on a patient who presented with nonspecific developmental delay and partial agenesis of the rostral corpus callosum. Fluorescence in situ hybridization (FISH) studies using probes specific for the PWS/AS region demonstrated a double signal on one chromosome 15, indicating the presence of an interstitial duplication of proximal 15q involving the PWS/ AS region in the patient. Parental chromosomes were normal with FISH studies. Methylation analysis at exon alpha of the SNRPN locus showed a maternal band at 4.2 kb and a paternal band of apparent double intensity at 0.9 kb, suggestive of one copy of the maternal allele and two copies of the paternal allele in the patient. Microsatellite analysis was informative at the GABRB3 locus in the family, which showed the inheritance of two different paternal alleles and a maternal allele in the patient consistent with the origin of this duplication from an unequal crossing over between the two chromosome 15 homologs in the father. This is the first report of an abnormal phenotype associated with a paternally derived duplication of proximal 15q shown to contain the PWS/AS region by molecular techniques.

Complex t(1;15;17) in acute promyelocytic leukemia with duplication of RAR alpha and PML sequences.

A 46-year-old white male presented with a two-week history of a flu-like illness and bleeding gums. A diagnosis of acute promyelocytic leukemia was made on bone marrow examination with accompanying DIC. All cytogenetically abnormal cells (28/30 at intake and 30/30 at two weeks post-induction) represented a single clone with apparent deletion of 1(p22) and 3(p25), and with a large, derivative chromosome 17. By conventional G- and C- banded analysis, the monocentric der(17) appeared to be disrupted distal to the typical (17q21) APL breakpoint, chromosome 15 did not demonstrate gross rearrangement, and the source of the additional material on the der(17) was unknown. Fluorescence in situ hybridization (FISH) with t(15;17), RAR alpha, and 17qter probes and with chromosome 1, 15, and 17 paints demonstrated that the der(17) consisted of a complex rearrangement with duplication of both RAR alpha and PML, insertion of chromosome 1 sequences, and double insertion of chromosome 15 sequences. The fusion of RAR alpha and PML consistent with APL appears to have occurred at the distal juxtaposition of these sequences in the derivative chromosome.

Telomere associations in desmoplastic infantile ganglioglioma.

A four and a half year old male was diagnosed with desmoplastic infantile ganglioglioma. To our knowledge, the cytogenetics of this tumor have never been reported. In our analysis of 40 cells, no consistent clonal abnormalities were observed; however, the majority of cells (25 of 40) showed structural rearrangements (telomere associations) resulting in dicentrics and other derivative chromosomes. Breakpoints most often observed included 17q25 (6 of 40), 19p13.3 (4 of 40), 17p13 (3 of 40), 14q32 (3 of 40), 11q25 (3 of 40), 9p24 (2 of 40), 5q35 (2 of 40), and 22q13 (2 of 40).

Constitutional de novo t(1;22)(p22;q11.2) and ependymoma.

There is a body of evidence suggesting the presence of a tumor suppressor gene on chromosome 22 which plays a role in the pathogenesis of ependymomas. We report a patient with a de novo constitutional t(1;22)(p22;q11.2) who developed a malignant ependymoma at age 5. The patient is otherwise phenotypically normal. By fluorescence in situ hybridization (FISH) analysis, the chromosome 22 breakpoint has been localized to the region between the DiGeorge locus and BCR. Since NF2 and EWS are both distal to BCR, the are presumable not involved in this rearrangement. This patient may offer a unique opportunity to identify the chromosome 22 ependymoma tumor suppressor gene by cloning the translocation breakpoint.

Diffuse large cell, B-cell type lymphoma with a novel translocation (2;22)(p23;q11.2).

We observed a translocation (2;22)(p23;q11.2) in the bone marrow cells of a patient with multiple subcutaneous nodules. Tumor histology and immunohistochemical staining demonstrated a malignant lymphoma, diffuse large cell type, displaying a CD30 negative B cell immunophenotype. To our knowledge, this is the first report of this specific translocation in lymphoma, which may join the site of the anaplastic lymphoma kinase (ALK) gene at 2p23 to the region of the immunoglobulin lambda light chain gene at 22q11.2. The ALK gene was initially identified through its involvement in the t(2;5)(p23;q35) found most commonly in anaplastic large cell lymphoma. This observation in a CD30 negative large cell lymphoma of B cell lineage further extends the relationship of anaplastic large cell morphology, ALK activation, lymphoid lineage, and expression of the CD30 antigen.

Amplification of the MLL region in acute myeloid leukemia.

We report amplification of the MLL gene region (11q23-->11qter) in a 72-year-old woman with myelodysplastic syndrome progressing to acute myelomonocytic leukemia and in a 51-year-old man with a history of hairy cell leukemia and secondary myelodysplasia progressing to acute myelogenous leukemia. The amplicons containing MLL were shown by molecular cytogenetics to extend from chromosomal region 11q23 to the distal long arm of chromosome 11 and to be present in the first patient in five copies on a large ring chromosome and present in the second patient also in five copies on two derived chromosomes. Other karyotypic findings in the first patient included del(5q), +8, and der(21)t(17;21), resulting in the loss of a copy of 17p, whereas deletion 7q was observed in the second patient. Southern-blot analysis for the second patient was consistent with MLL amplification but did not demonstrate rearrangement of the germ-line MLL band. Amplification of MLL and the 11q23 region has been documented in only a few cases and appears to be yet another mechanism by which MLL contributes to the leukemia phenotype.

Treatment of acromioclavicular separations. A retrospective study.

A retrospective study of 134 patients with Types I, II, and III acromioclavicular separations was carried out. The average followup was 6.3 years, with the longest being 19 years, and the shortest being 1 year. The mechanism of injury was a direct blow in 92% of the patients. The average age of the patients was 30.1 years, with a range from 13 to 68 years. All patients were evaluated using a standard rating system for the shoulder and humerus, the total for perfect recovery being 100. Twenty-four patients with Type I separations were immobilized 19.5 days, with a disability period of 6 weeks, and rated 94 points. Twenty-five patients with Type II separations were immobilized 27 days for the conservative groups, had a disability period of 6 weeks, and rated 90 points. Eighty-five patients with Type III acromioclavicular separations were followed. Seven patients had conservative treatment, were immobilized an average of 22 days, with a disability period of 13 weeks, and rated 82. Of those patients who underwent surgical repair, excluding Dacron graft substitution, the immobilization period was 6 weeks, with a disability period of 12 weeks, and a rating of 80. Fifty-eight patients underwent repair with double velour Dacron prosthetic substitution for the coracoclavicular ligaments, combined with distal clavicular resection in all but two patients. The average immobilization period was 1 week, with the average disability period being 3 weeks. The average rating was 96, with 24 patients rating 100. The major cause for a rating less than 100 was light to moderate pain that persisted in a few cases, which was only occasional and associated with a particular activity. One infection occurred requiring graft removal 5 months after surgery. Calcification in the area of the coracoclavicular ligaments did not affect the final rating and recurrence of deformity was not noted.

Natural history of anterior cruciate tears.

An evaluation of 361 patients with documented anterior cruciate tears was carried out with analysis of mechanisms, symptomatology, physical findings, and limitations. At the time of injury, patients generally heard a loud pop and felt their knee "slide apart" with a subsequent hemarthrosis. Internal tibial rotation was described as the principal mechanism of iniury in 81.6% of the patients. Less than 20% had a triad type injury. A "crossover" test enabled reproduction of the mechanism. On a 100 point scale, those with "no repair" scored a mean of 55.4, direct repairs 56.7, pes transfers 59.6, and modified MacIntosh 88.9 points. The natural course of a patient can often be projected. A Continuing comparative study of results from treatment regimens and procedures is indicated.

Extensive analysis of mosaicism in a case of Turner syndrome: the experience of 287 cytogenetic laboratories. College of American Pathologists/American College of Medical Genetics Cytogenetics Resource Committee.

OBJECTIVE: To assemble and interpret karyotype data provided as part of the College of American Pathologists/American College of Medical Genetics Cytogenetics Proficiency Testing Program. DATA SOURCES, EXTRACTION, AND SYNTHESIS: The Cytogenetics Resource Committee requested data on all cells analyzed in a 1994 whole-blood specimen challenge. In that study, 287 participating laboratories analyzed a total of 14297 cells derived from a sample drawn from an adult donor with Turner syndrome. This individual had previously been found to have mosaicism, including cell lines with X structural anomalies along with monosomy X, making this an excellent challenge for a multicenter cytogenetic survey. RESULTS AND CONCLUSIONS: Analysis of the data from this extensive study revealed mosaicism of up to 10 different sex chromosome complements involving the X chromosome with and without a small ring X or a derivative X chromosome. In the routine cytogenetic analysis performed by the participating laboratories, cell lines observed, in decreasing order of prevalence, included 45,X (n = 8357 cells), 46,X,r(X) (n = 3597), 46,X,der(X)t(X;X) (n = 2237), 46,XX (n = 93), 47,X,r(X),r(X) (n = 5), 47,X,der (X)t(X;X),der(X)t(X;X) (n = 3), 47,XX,r(X) (n = 2), and one observation each of 47,XX,der(X)t(X;X), 47,X,der(X)t (X;X),r(X), and 47,XXX. Our molecular cytogenetic data, as well as detailed analysis of G-banded chromosomes, suggest the nomenclature for these 2 abnormal X chromosomes as r(X)(p11.3q21.3) and der(X)t(X;X)(p11.3;q21.3), and we discuss models for the concomitant formation of these 2 entities. Both the degree of analysis and the extensive mosaicism that was discovered in this study are exceptional, and similar reported cases as well as possible mechanisms for the observed X chromosome instability are reviewed.