X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.     

Genetic complementation analysis of ataxia telangiectasia and Nijmegen breakage syndrome: a survey of 50 patients

Cultured cells from patients with ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS) are hypersensitive to ionizing radiation. After radiation exposure, the rate of DNA replication is inhibited to a lesser extent than in normal cells, whereas the frequency of chromosomal aberrations is enhanced. Both of these features have been used in genetic complementation studies on a limited series of patients. Here we report the results of extended complementation studies on fibroblast strains from 50 patients from widely different origins, using the radioresistant DNA replication characteristic as a marker. Six different genetic complementation groups were identified. Four of these, called AB, C, D, and E (of which AB is the largest), represent patients with clinical signs of AT. Patients having NBS fall into two groups, V1 and V2. An individual with clinical symptoms of both AT and NBS was found in group V2, indicating that the two disorders are closely related. In AT, any group-specific patterns with respect to clinical characteristics or ethnic origin were not apparent. In addition to the radiosensitive ATs, a separate category of patients exists, characterized by a relatively mild clinical course and weak radiosensitivity. It is concluded that a defect in one of at least six different genes may underlie inherited radiosensitivity in humans. To facilitate research on defined defects, a complete list of genetically characterized fibroblast strains is presented.     

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Summary Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several A-T-like genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open.     

Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: role in the initiation of sperm motility

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H-tetraphenylphosphonium (TPP) and 14C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.     

Structure and function of the mitochondrial bc1 complex. A mutational analysis of the yeast Rieske iron-sulfur protein

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to a single complementation group (G12) have been determined to have lesions in the iron-sulfur protein (Rieske protein) of ubiquinol: cytochrome c reductase. Mutants capable of expressing the protein were chosen for further studies. The genes from 13 independent isolates were cloned and their mutations sequenced. Twelve mutations were ascertained to cause single amino acid substitutions in the carboxyl-terminal regions of the protein between residues 127 and 173. This region is proposed to be part of the catalytic domain with the ligands responsible for co-ordinating the two irons of the 2Fe-2S cluster. Based on the catalytic properties of the ubiquinol: cytochrome c reductase complex and the electron paramagnetic resonance (e.p.r.) signals of the iron-sulfur protein, the mutants describe two different phenotypes. A subset of mutants have no detectable iron-sulfur cluster and are completely deficient in ubiquinol: cytochrome c reductase activity. These strains identify mutations in residues considered to be essential for binding of the iron or for maintaining a proper tertiary structure of the catalytic domain. A second group of mutants have reduced levels of enzymatic activity and exhibit e.p.r. spectra characteristic of the Rieske iron-sulfur cluster. The mutations in the latter strains have been ascribed to residues that influence the redox properties of the cluster by distorting the iron-binding pocket. A secondary and tertiary structure model is presented of the carboxyl-terminal 65 residues constituting the catalytic domain of the iron-sulfur protein. It is postulated that the two irons of the cluster are co-ordinated by three cysteine and a single histidine residue located in a loop structure. The catalytic domain also contains two short alpha-helices and three beta-strands that form a partial beta-barrel. Most of the hydrophilic amino acids are present in turns that map to one pole of the domain. When viewed in the context of the model, mutations that abolish the iron-sulfur cluster are mostly in residues defining the boundaries of the alpha-helices and beta-strands. The notable exception is a cysteine residue that has been assigned to the loop with the iron ligands. This cysteine residue is proposed to co-ordinate one iron of the cluster. Mutations that reduce ubiquinol: cytochrome c reductase activity and alter the redox potential of the cluster occur in residues located in the loop that contains the ligands of the cluster.     

Fine structure of the excretory system of the deep posterior (Ebner's) salivary glands of the human tongue

Human deep posterior lingual glands (von Ebner's glands) are located beneath the circumvallate papillae. They are formed by tubuloalveolar adenomeres, intercalated ducts and excretory ducts coming together in the main excretory duct. The tubuloalveolar cells, pyramid-shaped, show large and dense secretory granules (clear cored) throughout the cytoplasm, rare basal folds and packed cisternae of rough endoplasmic reticulum (RER) at the basal pole. The columnar cells of the intercalated ducts are arranged in a monolayer. They are characterized by dense, clear-core secretory granules (mostly in the apical cytoplasm), a basal nucleus, well-developed RER and Golgi apparatus, and thin filaments distributed in supra- and perinuclear cytoplasm. Striated ducts are absent. Excretory ducts, coming together in the main duct, are lined by a bistratified epithelium. The inner layer consists of columnar cells showing bundles of tonofilaments with scarce secretory activity. The outer layer is composed of basal cells lying on the basal lamina. The main excretory duct, which opens at the bottom of the vallum, shows a stratified epithelium. The outer side is composed of 2-3 layers of malpighian cells lying on the basal lamina. The inner side consists of a single layer of cuboidal-columnar cells with dense apical granules and well-developed organelles synthesizing and condensing secretions. These cells interpolate with goblet cells, rare mitochondria-rich cells, ciliated cells and numerous small globous cells showing a clear matrix and lacking secretory granules. The cilia show a 9 + 2 microtubular structure with basal bodies provided with striated rootlets. Myoepithelial cells surround with their processes the basal portions of the secretory cells and the intercalated ducts. The conclusions concern some comparative aspects and some hypothesis on the functional role of goblet cells, ciliated cells and epithelial cells lining the different ducts, also in relation to the final secretory product.     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

A high frequency of distinct ATM gene mutations in ataxia-telangiectasia.

The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families.     

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4, and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Nonsynaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4–7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they risulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/repefusion damage, showing changes earlier than the hippocampal ones.