Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.     

Immunologic characterization of CD7-deficient mice

Human CD7 is an Ig superfamily molecule that is expressed on mature T and NK lymphocytes. Although in vitro studies have suggested a role for CD7 in lymphoid development and function, the exact function of CD7 in vivo has remained elusive. One patient has been reported with SCID syndrome attributed to CD7 deficiency. To study in vivo functions of CD7, we have generated CD7-deficient mice and assessed their lymphoid development and function. CD7-deficient mice were viable, had normal peripheral blood and spleen lymphocyte numbers, and had normal specific Ab responses with Ag-driven Ig isotype switching. Thymocyte numbers were normal in 4-wk-old, 6-mo-old, and 1-yr-old CD7-deficient mice, but in 3-mo-old CD7-deficient mice, total thymocyte numbers were significantly increased by 60% (p < 0.02) compared with normal age-matched +/+ littermates. CD7-deficient splenocytes proliferated normally in response to various mitogens, including PHA, anti-CD3, Con A, and LPS. While NK cell numbers and cytolytic activity to YAC targets were normal, CD7-deficient mice had lower Ag-induced MHC class I-restricted CTL activity against OVA-transfected target cells than did +/+ control mice. Thus, CD7-deficient mice did not have a SCID syndrome, but rather had transient increases in thymocyte numbers at age 3 mo and altered splenocyte Ag-specific CTL effecter cell activity. These data suggest a role for CD7 in regulating intrathymic T cell development and in mediating CTL effecter function.     

Transcrystalline morphology of nylon 6 on the surface of aramid fibers

In this paper, the isothermal crystallization of nylon 6 in the presence of Kevlar 129 fiber was investigated by polarized optical microscopy (POM). The formation of a transcrystalline domain was found to be mainly controlled by crystallization conditions, such as the temperature of the isothermal crystallization, residual time at melting temperature and the cooling rate of the melt. The nucleation rate of nylon 6 on the fibers was mainly affected by the crystallization temperature. The interfacial transcrystallinity of nylon 6 occurred on the surface of Kevlar 129 fiber in the temperature range 130–190 °C. The reason for the formation of interfacial transcrystalline morphology is discussed from the molecular level, based on the understanding of the packing mode of nylon 6 chains around fibers and the interaction between matrix and fibers. It was found that the lattice matching and hydrogen‐bonding between nylon 6 and poly(p‐phenylene terephthalamide) (PPTA) crystals play an important role in the epitaxial crystallization. Copyright © 2004 Society of Chemical Industry     

Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay. The recombinant collagen binding domain of gelatinase A, comprised of the three fibronectin type II-like repeats, did not bind to TIMP-4, indicating that binding is mediated selectively by the C domain. Binding to TIMP-4 was of high affinity with an apparent Kd of 1.7 x 10(-7) M but slightly weaker than that to TIMP-2 (apparent Kd of 0.66 x 10(-7) M). Affinity chromatography confirmed the TIMP-4-C domain interaction and also showed that the complex could not be disrupted by 1 M NaCl or 10% dimethyl sulfoxide, thereby further demonstrating the tight binding. To verify the biological significance of this interaction, binding of full-length progelatinase A to TIMP-4 was investigated. TIMP-4 and TIMP-2 but not TIMP-1 bound specifically to purified TIMP-2-free human recombinant full-length progelatinase A and to full-length rat proenzyme from the conditioned culture medium of ROS 17/2.8 cells. Preincubation of the C domain with TIMP-2 was found to reduce subsequent binding to TIMP-4 in a concentration-dependent manner. Competition between TIMP-2 and TIMP-4 for a common or overlapping binding sites on the gelatinase A C domain may occur; alternatively TIMP-2 may prevent the binding of TIMP-4 by steric hindrance or induction of a conformational change in the C domain. We propose that the binding of progelatinase A to TIMP-4 represents a third TIMP-progelatinase interaction in addition to that of progelatinase A with TIMP-2 and progelatinase B with TIMP-1 described previously. This new phenomenon may be of important physiological significance in modulating the cell surface activation of progelatinase A.     

One-stage hypospadias repair with stent or suprapubic diversion: which is better?

PURPOSE: The authors report on 105 consecutive patients who underwent one-stage hypospadias repair based on use of suprapubic diversion or transurethral drainage with stenting. METHODS: The surgical procedures included 52 metal-based flap urethroplasty (Mathieu) for coronal, subcoronal, and distal shaft hypospadias; 32 transverse island pedicle graft (Duckett) for mid and proximal shaft hypospadias; 21 transverse island pedicle (Duckett) plus rolled midline tube (Thierchs) for penoscrotal and scrotal hypospadias. To accomplish urinary drainage, suprapubic diversion (cystofix) was used in 28 of 52 Mathieu operations, in 17 of 32 Duckett operations, and in 11 of 21 transverse island pedicle graft plus rolled midline tube operations. In the rest of the cases, transurethral drainage with stenting was used. RESULTS: All children had excellent cosmetic and functional outcomes. But the rates of complications such as fistula and meatal stenosis were significantly different between the groups in which suprapubic tube or urethral stent was used. In 56 of the 105 patients in whom suprapubic diversion was used, four (7.14%) had fistulas and three (5.35%) had meatal stenosis, in contrast to a fistula rate of 14.28% and meatal stenosis rate of 12.24% in patients that urethral stent is used for urinary drainage. CONCLUSION: The authors believe that the use of suprapubic diversion is advantageous for the outcome of one-stage hypospadias repair in relation to fistula occurrence and meatal stenosis.     

Hair analysis does not support hypothesized arsenic and chromium exposure from drinking water in Woburn, Massachusetts

We hypothesized that residents of Woburn, Massachusetts, had been exposed to as much as 70 microg/l of arsenic (As) and 240 microg/l of chromium (Cr) in drinking water from municipal supply wells G and H. To test this hypothesis, we measured the concentrations of As and Cr in 82 hair samples donated by 56 Woburn residents. Thirty-six samples were cut between 1964 and 1979, the period during which wells G and H were in operation. The remainder were cut either before 1964 (1938-1963; n = 26) or after 1979 (1982-1994; n = 20). Washed hair samples were analyzed by instrumental neutron activation. Exposure to the well water--measured as access--was estimated using well pumping records and a model of the Woburn water distribution system. Our results show that access to wells G and H water was not significantly correlated (95% confidence interval) with As and Cr concentrations measured in the hair of Woburn residents, but As concentrations have declined significantly over the last half century. Linear regression of As concentrations (micrograms per gram) upon year of hair cut and access to wells G and H water yielded a standard coefficient for year of -0. 0074 +/- 0.0017 (standard error; p = 2.5 -multiple- 10(-5)) and -0.12 +/- 0.10 (p = 0.22) for access. The r2 value for the model was 0.19. The geometric mean concentrations (geometric standard deviation) of As and Cr in the hair of residents who had access (i.e., relative access estimate >0) to wells G and H water (n = 27) were 0.14 (2.6) and 2.29 (1.8) microg/g, respectively; the geometric mean concentrations of As and Cr in all of the hair samples from residents who did not have access (1938-1994; n = 55) were 0.13 (3.0) and 2.19 (2.0) microg/g, respectively.     

Requirement of Drosophila NF1 for activation of adenylyl cyclase by PACAP38-like neuropeptides

The human neurofibromatosis type 1 (NF1) tumor suppressor protein functions as a Ras-specific guanosine triphosphatase-activating protein, but the identity of Ras- mediated pathways modulated by NF1 remains unknown. A study of Drosophila NF1 mutants revealed that NF1 is essential for the cellular response to the neuropeptide PACAP38 (pituitary adenylyl cyclase-activating polypeptide) at the neuromuscular junction. The peptide induced a 100-fold enhancement of potassium currents by activating the Ras-Raf and adenylyl cyclase-adenosine 3',5'-monophosphate (cAMP) pathways. This response was eliminated in NF1 mutants. NF1 appears to regulate the rutabaga-encoded adenylyl cyclase rather than the Ras-Raf pathway. Moreover, the NF1 defect was rescued by the exposure of cells to pharmacological treatment that increased concentrations of cAMP.