Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

Translational regulation of somatostatin in cultured sympathetic neurons

Coculture of sympathetic neurons with ganglion nonneuronal cells elevated levels of preprosomatostatin mRNA but did not alter neuronal synthesis, content, or release of somatostatin. Treatment of sympathetic neurons with culture medium conditioned by exposure to ganglion nonneuronal cells similarly elevated preprosomatostatin mRNA. Treatment with conditioned medium elevated somatostatin levels in pure neuronal cultures, but not in neurons cocultured with nonneuronal cells. Conditioned medium also failed to increase peptide levels in neurons cultured on a substratum of killed nonneuronal cells, despite a large increase in preprosomatostatin mRNA. These observations suggest that contact of sympathetic neurons with nonneuronal cell membranes inhibits the increase in peptide synthesis, but not the increase in preprosomatostatin mRNA after treatment with conditioned medium. Thus neuronal interactions with nonneuronal cells regulate somatostatin metabolism at both the mRNA and peptide levels. Regulatory effects on the mRNA and the peptide are separable and do not necessarily occur in parallel, and translational controls may be the rate-limiting factors.     

The GLI-Kruppel family of human genes.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.     

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.     

A differential medium for the identification of races 1 and 4 of Fusarium oxysporum f.sp. cubense

Three distinctive colony types were produced when Fusarium oxysporum f.sp. cubense (Foc ) races 1 and 4 were cultured on a defined basal medium containing an appropriate carbon source and bromothymol blue as a pH indicator. These distinctive cultural characteristics have been used as a specific and reliable method for the differentiation of races 1 and 4 of Foc from other species of Fusarium.     

Recognition of general patterns using neural networks

The Hopfield model of neural network stores memory in its symmetric synaptic connections and can only learn to recognize sets of nearly orthogonal patterns. A new algorithm is put forth to permit the recognition of general (non-orthogonal) patterns. The algorithm specifies the construction of the new network's memory matrix T _ij, which is, in general, asymmetrical and contains the Hopfield neural network (Hopfield 1982) as a special case. We find further that in addition to this new algorithm for general pattern recognition, there exists in fact a large class of T _ij memory matrices which permit the recognition of non-orthogonal patterns. The general form of this class of T _ij memory matrix is presented, and the projection matrix neural network (Personnaz et al. 1985) is found as a special case of this general form. This general form of memory matrix extends the library of memory matrices which allow a neural network to recognize non-orthogonal patterns. A neural network which followed this general form of memory matrix was modeled on a computer and successfully recognized a set of non-orthogonal patterns. The new network also showed a tolerance for altered and incomplete data. Through this new method, general patterns may be taught to the neural network.     

Analysis for linkage between F13A and three chromosome 6 marker loci: evidence for 6pter: F13A:HLA:GL01:cen gene order

Summary The results of the present study provide independent support for F13A:HLA linkage and refine the F13A: HLA and F13A: GLO1 linkage relationships. Analysis of the corresponding recombination fractions for the total paternal F13A:HLA and F13A:GLO1 peak lod scores() indicates a locus order of 6pter: F13A:HLA:GLO1:cen. Lod scores between F13A and PLG, a locus recently assigned to chromosome 6, exclude close linkage between these loci.     

The molecular mechanism of the bicarbonate effect at the plastoquinone reductase site of photosynthesis

It has been known for some time that bicarbonate reverses the inhibition, by formate under HCO₃ ^--depletion conditions, of electron transport in thylakoid membranes. It has been shown that the major effect is on the electron acceptor side of photosystem II, at the site of plastoquinone reduction. After presenting a historical introduction, and a minireview of the bicarbonate effect, we present a hypothesis on how HCO₃ ^- functions in vivo as (a) a proton donor to the plastoquinone reductase site in the D1-D2 protein; and (b) a ligand to Fe²⁺ in the Q_A-Fe-Q_B complex that keeps the D1-D2 proteins in their proper functional conformation. They key points of the hypothesis are: (1) HCO₃ ^- forms a salt bridge between Fe²⁺ and the D2 protein. The carboxyl group of HCO₃ ^- is a bidentate ligand to Fe²⁺, while the hydroxyl group H-bonds to a protein residue. (2) A second HCO₃ ^- is involved in protonating a histidine near the Q_B site to stabilize the negative charge on Q_B. HCO₃ ^- provides a rapidly available source of H⁺ for this purpose. (3) After donation of a H⁺, CO₃ ^2- is replaced by another HCO₃ ^-. The high pKa of CO₃ ^2- ensures rapid reprotonation from the bulk phase. (4) An intramembrane pool of HCO₃ ^- is in equilibrium with a large number of low affinity sites. This pool is a H⁺ buffering domain functionally connecting the external bulk phase with the quinones. The low affinity sites buffer the intrathylakoid [HCO₃ ^-] against fluctuations in the intracellular CO₂. (5) Low pH and high ionic strength are suggested to disrupt the HCO₃ ^- salt bridge between Fe²⁺ and D₂. The resulting conformational change exposes the intramembrane HCO₃ ^- pool and low affinity sites to the bulk phase.Two contrasting hypotheses for the action of formate are: (a) it functions to remove bicarbonate, and the low electron transport left in such samples is due to the left-over (or endogenous) bicarbonate in the system; or (b) bicarbonate is less of an inhibitor and so appears to relieve the inhibition by formate. Hypothesis (a) implies that HCO₃ ^- is an essential requirement for electron transport through the plastoquinones (bound plastoquinones Q_A and Q_B and the plastoquinone pool) of photosystem II. Hypothesis (b) implies that HCO₃ ^- does not play any significant role in vivo. Our conclusion is that hypothesis (a) is correct and HCO₃ ^- is an essential requirement for electron transport on the electron acceptor side of PS II. This is based on several observations: (i) since HCO₃ ^-, not CO₂, is the active species involved (Blubaugh and Govindjee 1986), the calculated concentration of this species (220 M at pH 8, pH of the stroma) is much higher than the calculated dissociation constant (Kd) of 35–60 M; thus, the likelihood of bound HCO₃ ^- in ambient air is high; (ii) studies on HCO₃ ^- effect in thylakoid samples with different chlorophyll concentrations suggest that the left-over (or endogenous) electron flow in bicarbonate-depleted chloroplasts is due to left-over (or endogenous) HCO₃ ^- remaining bound to the system (Blubaugh 1987).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (common name: diuron) - PSII photosystem II - Q_A first plastoquinone electron acceptor of PSII - Q_B second plastoquinone acceptor of PS II