A note on dimensionless life histories for birds versus mammals

Summary Offspring production over the adult lifespan (b/M whereb is the yearly fledgling or offspring production and 1/M is the mean adult lifespan) is an approximate invariant within both birds and mammals. The two taxa differ, however, in that mammals have bothM and b as invariants (b/M = b/M) while birds do not ( is the age at first breeding). Birds have a surprising cancellation in that bothM andb are ^–0.25.     

Nucleotide sequence of a DNA region comprising the gene for elongation factor 1α (EF-1α) from the ultrathermophilic archaeotePyrococcus woesei: Phylogenetic implications

Summary The gene encoding elongation factor 1 (EF-1, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeotePyrococcus woesei was localized within aBglII fragment of chromosomal DNA. Sequence analysis showed that the EF-1 gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNA_ser (GGA). The three genes follow each other immediately in the order EF-1·S10·tRNA_ser after a putative promoter located 55 bp upstream of the EF-1 gene. Alignment of the derived EF-1 sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed thatPyrococcus EF-1 is highly homologous (89% identity) toThermococcus celer EF-1, both being strikingly more similar to eukaryotic EF-1 than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony-although not evolutionary parsimony-support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of thePyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation ofPyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.     

Molecular Genetic Analysis of the Interleukin 6 and Tumor Necrosis Factor α Gene Polymorphisms in Multiple Myeloma

The –174G C polymorphism of the interleukin 6 (IL-6) gene promoter and the –308G A polymorphism of the tumor necrosis factor (TNF) gene promoter were tested for association with multiple myeloma (MM) varying in severity. Of 69 patients, 19 had aggressive MM, 26 had benign MM, and 24 had unidentified MM. The control group (N = 102) matched the test group in age and sex composition. The two groups did not significantly differ in allele and genotype frequencies of the IL-6 and TNF genes. Genotype CC, which determines low-level expression of IL-6, occurred at a frequency of 0.35 in patients with low-progressing MM and was absent from patients with aggressive MM. The TNF gene showed no association with predisposition to MM or clinical variant of the disease. As for MM progression, genotype CC of the IL-6 gene was associated with milder clinical signs in patients from Bashkortostan.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Genetic polymorphisms of gene conversion within the duplicated human α-globin loci

Summary Of the 645 Japanese subjects studied, we have identified 10 individuals heterozygous for a chromosome with the triplicated -globin loci. The frequency of the triple -loci was 0.008 in this population, while that of the single -locus, i.e., -thalassemia 2 gene, might be lower than 0.0008. Analysis of haplotypes using particular RsaI site polymorphism in the -globin gene complex strongly suggests that the triple loci may have had multiple origins in this population.     

Subunit Interacting Surfaces of Human Hemoglobin in Solution: Localization of the α–β Subunit Interacting Surfaces on the α-Chain by a Comprehensive Synthetic Strategy

By using synthetic overlapping peptides encompassing the entire -chain of adult human hemoglobin (HbA), we have mapped on the -chain the regions responsible for its binding to the -chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides 81–95, 101–115, 111–125, and 131–141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide 31–45, which in the crystal had the highest number of contact residues of all the -chain peptides, did not bind the -chain in solution. Similarly, peptide 91–105, with seven contact residues in the crystal, showed low binding with the -chain in solution. On the other hand, peptides 41–55 and 121–135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide 121–135 had the highest binding activity of the -chain peptides. These studies and our previous findings, which localized on the -chain the regions that bind to the -chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.     

Cytokines in Brain Ischemia—The Role of TNFα

1. The role of cytokines and other inflammatory mediators in the progression of ischemic brain injury is a new and exciting era of research. Evidence in support for a role for TNF in this respect is emerging as evidence on de novo upregulation of TNF following ischemia is now well established.2. TNF administered directly to the brain parenchyma elicits local microvascular injury in the form of pericapillary edema and leukocyte adhesion to cerebral capillaries.3. TNF administered into the cerebroventricular space prior to ischemia augment the extent of tissue damage and neurological deficits.4. Specific and potent inhibitors of TNF synthesis or TNF receptors must be developed and tried to prove firmly a role for TNF in ischemic brain injury.     

Energy-transducing nicotinamide nucleotide transhydrogenase: Nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme fromRhodospirillum rubrum

Based on the amino acid sequence of the N-terminus of the soluble subunit of theRhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4kbp was isolated from a genomic library ofR. rubrum and sequenced. This sequence contained three open reading frames, constituting the genesnntA1, nntA2, andnntB of theR. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated 1, 2, and , respectively, and are considered to be the subunits of theR. rubrum transhydrogenase. The predicted amino acid sequence of the 1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the subunit of theEscherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a fold in the corresponding region, and the purified, soluble 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the subunit of theR. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the subunit of theE. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores ofR. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the 2 subunit of theR. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the subunit of theE. coli transhydrogenases.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

Effects of transforming growth factor β, tumor necrosis factor α and interferon γ on pancreatic islet β-cell responsiveness to transforming growth factor α

The insulin-producing pancreatic islet -cell, characterized by low proliferative potential, is normally not responsive to the polypeptide epidermal growth factor (EGF) or its homolog transforming growth factor (TGF-). Since EGF receptors in other tissues can be up-regulated by other growth factors and by cytokines, we have in this paper investigated whether such a -cell responsiveness to TGF-, or EGF, can be conferred by co-culture with interferon (IFN-), tumor necrosis factor (TNF-) or transforming growth factor (TGF-) in various combinations. To this end, fetal rat pancreatic islets enriched in -cells were isolated and cultured for 3 days with or without 200 pM or 20 nM TGF-. It was found that neither of these TGF- concentrations affected -cell mitogenesis, insulin content or insulin secretion. However, IFN- (1000 U/ml) evoked a modest stimulation of -cell replication, while suppressing insulin secretion and leaving the islet insulin content unaltered. TNF- (1000 U/ml), on the other hand, affected none of these parameters either alone or in any combination with TGF- or IFN-. However, when TNF- or IFN-, either alone or in combination, were combined with the cytokine interleukin-1, this resulted in islet disintegration, whereas the latter cytokine alone did not exert any gross necrotic changes evident by light microscopy. TGF- (500 pM) stimulated insulin secretion but did not influence islet insulin content or -cell mitogenesis either alone or in combination with TGF- (200 pM or 20 nM). In no instance could any mitogenic or secretory response to low or high concentrations of TGF- be conferred by IFN-, TNF- or TGF- whether used alone or in combinations. Hence, responsiveness to TGF- or EGF in the -cell obviously cannot be achieved by any of these peptides.Abbreviations EGF epidermal growth factor - IFN- interferon - TGF- transforming growth factor - TGF- transforming growth factor - TNF- tumor necrosis factor      

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

Effect of chronic desipramine treatment on neurotransmitter-thyrotropin releasing hormone—thyrotropin interactions in the rat

Long-term administration of the antidepressant drug, desipramine (20 mg/kg/day, orally for 28 days), decreased the stimulatory effect of the ₂-adrenoceptor agonist, clonidine (250 g/kg, i.p.) on thyrotropin (TSH) secretion in the rat, but did not alter basal TSH secretion. -Adrenoceptor-mediated inhibition of TSH secretion by isoproterenol (1 mg/ kg, i.p.) was unaffected by chronic desipramine treatment, as were the stimulatory effect of TSH-releasing hormone (TRH, 5 g/kg, i.v.) on TSH release and its inhibition by the -adrenoceptor antagonist, phentolamine (2 mg/kg, i.p.). These findings suggest that chronic desipramine treatment induces subsensitivity of ₂-adrenoceptors which modulate TSH secretion in the rat while not affecting -adrenoceptor-mediated inhibition of TSH release. These findings suggest that pituitary TRH receptors are unchanged but that changes occurred at the hypothalamic level in ₂-adrenoceptor-mediated stimulation of TRH release. Although cerebral -adrenoceptors have been shown convincingly to be down-regulated after chronic desipramine treatment, their function in the hypothalamic TRH system after 28 days of treatment with desipramine appears to be unimpaired.     

Identification of surface exposed amino acids of isolated and membrane-bound CF1 by protease accessibility

In order to compare surface-exposed amino acids in isolated and membrane-bound CF₁ the technique of limited proteolysis was employed. The cleavage sites of several proteases were identified by sequence analysis of the resulting peptides after isolation by SDS-PAGE. In isolated CF₁ the N-terminal region of the subunit was found to be highy sensitive to proteases; the accessible peptide bonds included E17-G18, R21-E22, E22-V23, and K24-V25. Additional protease-attacked bonds in subunit were S86-S87, xE125-S126. and R127-L128. In the subunit of isolated CF₁ the bonds L14-E15 and V76-A77 were identified as being accessible. All identified protease accessible amino acids are located at the protein surface according to a molecular model of CF₁ computed after the crystal structure of mitochondrial F₁ by S. Engelbrecht (1997). In membrane bound CF₁ the primarily accessible peptide bond of the N-terminal domain of is R21-E22. After this bond is cleaved by trypsin, the K24-V25 becomes accessible to further trypsin attack. Moreover, the peptide bonds R14O-S141 and G16O-R161 are cleaved. According to the Engelbrecht model G16O is almost completely shielded and actually this amino acid was hardly accessible to protease in isolated CF₁. The subunit in general is much more sensitive to proteolysis in membrane-bound than in solubilized CF₁. In the subunit of membrane-bound CF₁ a papain-sensitive bond G102-G103 was identified. The results indicate major structural alterations when CF₁ is extracted from the CF₀CF₁ complex. Thiol modulation, i.e. reduction of the regulatory disulfide bond between C199 and C205 of y subunit, enhances the accesibility of a number of peptide bonds, in particular G160-R161, to proteolytic attack by papain. In contrast, thylakoid membrane energization results in masking of this peptide bond.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.