Nucleotide sequence evidence of the unicentric origin of the βC mutation in Africa

Summary The origin of the ^C mutation was studied by characterizing nucleotide sequence polymorphisms on ^C chromosomes of patients from various African countries. In the majority of cases, the ^C mutation was found in linkage disequilibrium with a single chromosomal structure as defined by classical RFLP haplotypes, intergenic nucleotide sequence polymorphisms immediately upstream of the -globin gene, and intragenic -globin gene polymorphisms (frameworks). In addition, three atypical variant chromosomes carrying the ^C mutation were observed, and are most probably explained either by a meiotic recombination (two cases) or by one nucleotide substitution occurring in an unstable array of tandemly repeated sequences (one case). These data demonstrate the unicentric origin of the ^C mutation in central West Africa, with subsequent mutational modification in a small number of instances. The data also supports gene flow of the ^C chromosome from subsaharan Africa to North Africa.     

3H-thymidine incorporation at the end of the S phase in cultured human lymphocytes

Summary Cultured human lymphocytes of female origin were exposed to 3 Ci/ml ³H-thymidine for 2, 21/4, 21/2, 23/4, and 3 hrs, respectively; a proportion of 0, 1.95, 6.4 and 16.7% of labeled metaphases were found among a total of over 3000 metaphases examined. 25 metaphases with little overall radioactivity were analyzed in detail by grain count and grain distribution. Earlier studies were confirmed that the grain distribution at the extreme end of the S phase is non-random in several regions of the chromosomal complement, but that a distinct interval (Z interval) probably does not exist.

---

Zusammenfassung In der vorliegenden Arbeit wird das Ende der DNA-Synthese mittels autoradiographischer Methoden an normalen menschlichen Lymphocyten untersucht. Das Reduplikationsverhalten der Chromosomen am Ende der DNA-Synthese wird näher beschrieben und diskutiert.Das Reduplikationsmuster der letzten 30 min der S-Phase als auch das Verhalten des späten X-Chromosoms sowie der Vergleich zum Markierungsverhalten der Autosomen sprechen gegen die Existenz eines sogenannten Z-Intervalls am Ende der Synthese-Phase.

---

---

This investigation was supported in part by grants from the Deutsche Forschungsgemeinschaft and is part of a thesis by D. B. in fulfillment of requirements for the M. D., at the University of Hamburg.     

Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized beta-galactosidase from Kluyveromyces lactis

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free -galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the -galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized -glucosidase and with added co-solvents such as hexanol.     

Progressively developed myocardial apoptotic cell death during late phase of reperfusion

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins and infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flow, measured by colored microspheres during ischemia. Necrotic cell death (TTC staining) was significantly increased during R, starting at 27 ± 2% at 6 h R and increasing to 41 ± 2% at 24 h R. There was no further change at 48 (37 ± 3%) and 72 (36 ± 6%) h R, respectively. TUNEL positive cells (% total normal nuclei) in the peri-necrotic zone progressively increased from 6 (26 ± 2^*) to 24 (38 ± 1^*), 48 (48 ± 3^*) and 72 (59 ± 4^*) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compared with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 ± 0.8%^*) to 72 h R (78 ± 2%^*), and increase in expression of pro-apoptotic proteins including Bax from 6 (30 ± 3%^*) to 72 h R (66 ± 3%^*), and p53 from 6 (12 ± 1%^*) to 72 h R (91 ± 2%^*), respectively. Immunohistochemical staining revealed that infiltrated neutrophils (mm² myocardium) were significantly correlated with development of necrotic and apoptotic cell death from 6 to 24 h R, respectively (P < 0.05), while large macrophage infiltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apoptosis; 3) inflammatory cells may play a role in triggering cell death during R. ^* P < 0.05 vs. normal nuclei and tissue; P < 0.01 vs. 6 h R.     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Environmental Estrogen-Like Chemicals and Hydroxyl Radicals Induced by MPTP in the Striatum: A Review

Oxygen free radical formation has been implicated in lesions caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP⁺) by type B monoamine oxidase (MAO) in the brain, the etiology of this disease remains obscure. This review focuses on the role of an environmental neurotoxin chemically related to MPP⁺-induced free radical generation in the pathogenesis of Parkinson's disease. Environmental-like chemicals, such as para-nonylphenol or bisphenol A, significantly stimulated hydroxyl radical (OH) formation in the striatum. Allopurinol, a xanthine oxidase inhibitor, prevents para-nonylphenol and MPP⁺-induced OH generation. Tamoxifen, a synthetic nonsteroidal antiestrogen, suppressed the OH generation via dopamine efflux induced by MPP⁺. These results confirm that free radical production might make a major contribution at certain stages in the progression of the injury. Such findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson's disease and traumatic brain injuries.     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

Effects of different secretagogues and intracellular messengers on the muscarinic modulation of [3H]acetylcholine release

We have investigated whether muscarinic receptors modulate the release of [³H]ACh elicited by secretagogues that act by different mechanisms in rat cerebral cortical synaptosomes. Oxotremorine (10 M) reduced the calcium-dependent [³H]ACh release induced by mild K⁺-depolarization (10 and 15 mM K⁺), but not that by higher K⁺ concentrations. The ACh-release induced by A23187 (0.2–5 g/ml), liposomes laden with 113 mM CaCl₂, or 4-aminopyridine (1–10 mM) was not modulated by oxotremorine. Ouabain (100 M)-induced release of [³H]ACh was reduced by oxotremorine in normal but not calcium-free KR, indicating that extracellular calcium-uptake but not Na⁺, K⁺-ATPase activity may be necessary for release-modulation. With respect to possible second messenger systems, dibutyrylcyclic AMP (0.1–2 mM), dibutyrylcyclic GMP (0.1–2 mM), forskolin (100 M), and phorbol ester (0.3–3 g/ml) were without effect on release or release-modulation. These results are consistent with an involvement of K⁺-channels and voltage-sensitive calcium-channels in the muscarinic release-inhibition process. They argue against an involvement of Na⁺, K⁺-ATPase, adenylate cyclase, guanylate cyclase, and phosphatidylinositol turnover in the release-modulation process.     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Plasma membrane was purified from roots of rye (Secale cereale L. cv. Rheidol) by aqueous-polymer two-phase partitioning and incorporated into planar bilayers of 1-palmitoyl-2-oleoyl phosphatidylethanolamine by stirring with an osmotic gradient. Since plasmamembrane vesicles were predominantly oriented with their cytoplasmic face internal, when fused to the bilayer the cytoplasmic side of channels faced the trans chamber. In asymmetrical (cis:trans) 280100 mM KCl, five distinct K⁺-selective channels were detected with mean chord-conductances (between +30 and -30 mV; volyages cis with respect to trans) of 500 pS, 194 pS, 49 pS, 21 pS and 10 pS. The frequencies of incorporation of these K⁺ channels into the bilayer were 48, 21, 50, 10 and 9%, in the order given (data from 159 bilayers). Only the 49 pS channel was characterized further in this paper, but the remarkable diversity of K⁺ channels found in this preparation is noteworthy and is the subject of further study. In symmetrical KCl solutions, the 49 pS channel exhibited non-ohmic unitary-current/voltage relationships. The chord-conductance (between +30 and-30 mV) of the channel in symmetrical 100 mM KCl was 39 pS. The unitary current was greater at positive voltages than at corresponding negative voltages and showed considerable rectification with increasing positive and negative voltages. This would represent inward rectification in vivo. Gating of the channel was not voltage-dependent and the channel was open for approx. 80% of the time. Presumably this is not the case in vivo, but we are at present uncertain of the in vivo controls of channel gating. The distribution of channel-open times could be approximated by the sum of two negative exponential functions, yielding two open-state time constants (_o, the apparent mean lifetime of the channel-open state) of 1.0 ms and 5.7 s. The distribution of channel-closed times was best approximated by the sum of three negative exponential functions, yielding time constants (_c, the apparent mean lifetime of the channel-closed state) of 1.1 ms, 51 ms and 11 s. This indicates at least a five-state kinetic model for the activity of the channel. The selectivity of the 49 pS channel, determined from both reversal potentials under biionic conditions (100 mM KCl100 mM cation chloride) and from conductance measurements in symmetrical 100 mM cation chloride, was Rb⁺ K⁺ > Cs⁺ > Na⁺ > Li⁺ > tetraethylammonium (TEA⁺). The 49 pS channel was reversibly inhibited by quinine (1 mM) but TEA⁺ (10 mM), Ba²⁺ (3 mM), Ca²⁺ (1 mM), 4-aminopyridine (1 mM) and charybdotoxin (3 M) were without effect when applied to the extracellular (cis) surface.Abbreviations and Symbols GHK Goldman-Hodgkin-Katz - I/V current/voltage - PEG polyethyleneglycol - P_o probability o f the channel being open - TEA⁺ tetraethylammonium - _c apparent mean lifetime of the channel-closed state - _o apparent mean lifetime of the channel-open state P.J.W. was supported by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Professor E.A.C. MacRobbie and M.T. by the Glaxo Junior Research Fellowship at Churchill College, Cambridge. We thank Dr. D.T. Cooke (AFRC, Long Ashton Research Station, University of Bristol, UK) and Ms. J. Marshall (University of York, UK) for their advice and assistance with the aqueous-polymer two-phase partitioning of plasma membrane from rye roots, Mr. J. Banfield and Miss P. Parmar (University of Cambridge, UK) for technical assistance and Professor E.A.C. MacRobbie, Dr. G. Thiel (University of Cambridge, UK), Dr. M.R. Blatt (Wye College, University of London, UK), Dr. D. Sanders and Dr. E. Johannes (University of York, UK) for helpful discussions.     

Biotransformation of cholesterol by Micrococcus species mediated by plasmid DNA

A steroid-biotransforming strain RJ6 was identified as Micrococcus roseus. This bacterium has a 10kb plasmid pMQV10. Curing mediated through cultivation of the culture with a low concentration (200ng/ml) of mitomycin C is described. Loss of cholesterol degradation (chol⁺) and streptomycin resistance (Sm^r) phenotypes as a consequence of the loss of plasmid indicate the extrachromosomal location of these two genes in this strain. An electroporation procedure was developed for transformation of cured strain of Micrococcus (RJC6) by plasmids. Frequency of greater than 10⁵ transformants/g DNA was achieved, which is 100-fold higher than the standard transformation procedure that yielded 5.3×10³ transformants/g DNA in the same strain.     

Glucosylation of cyclodextrin-complexed podophyllotoxin by cell cultures of Linum flavum L.

The glucosylation of the cytotoxic lignan podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, dimethyl--cyclodextrin and hydroxypropyl--cyclodextrin were used to improve the solubility of podophyllotoxin by complexation. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM, using a podophyllotoxin/cyclodextrin ratio of 1:1. Growth parameters of the cell suspensions were not affected neither by the addition of cyclodextrins alone, nor when complexed podophyllotoxin was dissolved in the medium.The complexed lignan disappeared rapidly from the culture medium, within 24h, under all experimental conditions. Almost simultaneously, between 73 and 100% of detectable podophyllotoxin was bioconverted into podophyllotoxin--d-glucoside. A maximal bioconversion rate of 0.51 mmol l^-1 suspension day^-1 was calculated for the L. flavum cells growing in a medium which included the podophyllotoxin/dimethyl--cyclodextrin complex at a final concentration of 1.35 mM.     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

The levels of ζ, γ, and δ chains in patients with Hb H disease

Summary Details are given of a study of blood samples from 24 patients with Hb H disease from different Mediterranean countries and from the Far East. Four different types of -thal-1 (--) were observed, namely-() ( 20.5-kb deletion);--MED-I ( 17.5-kb deletion);--MED-II (>26.5-kb deletion); and--SEA ( 18-kb deletion, in Orientals only). The -thal-2 was mainly of the deletion type (16 with the 3.7-kb deletion; 1 with the 4.2-kb deletion), while 4 of the 7 patients with a nondeletional type had the five-nucleotide deletion at the donor splice site of the first intron of the 2 gene. All patients had a mild-to-moderate hemolytic anemia; no significant differences in hematology were observed between the groups. Hb A₂ was decreased to about one-third of the normal level. The Hb H formation varied considerably and its quantitation was not always satisfactory. Patients with Hb H disease due to any -thal-1 combined with a nondeletional -thal-2 had the highest Hb H levels and a more marked anemia. The chain production was small and absent in patients with the MED-II type of -thal-1 because this deletion included the and genes. The highest chain levels were present in the four patients with the SEA type of -thal-1. The chain production was increased, particularly in patients with a mutation of C T at position-158 to the ^G globin gene. This chain was primarily present as Hb Bart's (or ₄) and only about 15% was recovered as Hb F or ₂₂. The evaluation of the rate of chains produced in these patients was greatly facilitated by data from one patient who had Hb H disease and a heterozygosity for the ^A-⁺. The low levels of Hb A₂ and of Hb F (relative to Hb Bart's) can be explained by a decreased affinity of chains for and chains as compared with chains in conditions of severe chain deficiency.     

Peptidergic neurons of the crab,Cardisoma carnifex,in defined culture maintain characteristic morphologies under a variety of conditions

Summary Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, superlarge, found occasionally, has a soma diameter greater than 40 m and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with l-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26° C instead of 22° C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K⁺]_o medium ([K⁺]_o=15–110 mM; standard=11 mM) has no long-term effect, but growth is arrested by [K⁺]_o greater than 30 mM. Cultures were also grown in media in which [Ca²⁺]_o ranged from 0.1 M to 26 mM (standard=13 mM). Outgrowth occured from all neuronal types in all [Ca²⁺]_o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV