Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

An Immunological Assay for Detection and Enumeration of Thermophilic Biomining Microorganisms

A specific, fast, and sensitive nonradioactive immunobinding assay for the detection and enumeration of the moderate thermophile Thiobacillus caldus and the thermophilic archaeon Sulfolobus acidocaldarius was developed. It employs enhanced chemiluminescence or peroxidase-conjugated immunoglobulins in a dot or slot blotting system and is very convenient for monitoring thermophilic bioleaching microorganisms in effluents from industrial bioleaching processes.     

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.     

Vanadium compounds

The direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity was investigated. Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and from bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALP-activity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP.     

Analysis by Polymerase Chain Reaction of α1 and α6 GABAA Receptor Subunit mRNAs in Individual Cerebellar Neurons After Whole-Cell Recordings

Abstract: With the use of the single-cell polymerase chain reaction (PCR), the GABA_A receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to α1 and α6 GABA_A receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, α1 subunit mRNA but not α6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the α1 and α6 GABA_A receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of α6 subunit mRNA.     

Larval development of three blenniid species Aidablennius sphynx, Coryphoblennius galerita and Lipophrys canevai (Pisces: Blenniidae: Blenniini) in the western Mediterranean

The early stages of development of three blenniid species, Aidablennius sphynx (6.7–15.8 mm BL), Coryphoblennius galerita (4.3–13.9mm BL), and Lipophrys canevai (3.5–10.4 mm BL) are described from specimens collected in the western Mediterranean. The characteristics used for identification included meristic, developmental, morphological and pigmentation characters. Distinguishing characters of these species useful in differentiating them from other species of blenniids for which early stages are known in the Mediterranean are presented. Information on the occurrence of larvae and juveniles of these species off the Catalan coast (north-western Mediterranean) is also given.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Sensorimotor transformation from light reception to phototactic behavior inDrosophila larvae (Diptera: Drosophilidae)

In this paper we examine theDrosophila melanogaster larval response to light. We survey the morphology of the larval visual and motor systems in relation to larval locomotory behavior and phototaxis. In addition, this paper proposes a model of sensorimotor transformation and examines the reversal in taxis occurring at theD. melanogaster larval wnadering stage.