1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Markov model for analysing cancer markers and disease states in survival studies

In studies of serial cancer markers or disease states and their relation to survival, data on the marker or state are usually obtained at infrequent time points during follow-up. A Markov model is developed to assess the dependence of risk of death on marker level or disease state and inferences within this model are based directly on data collected in this haphazard way. An application relating changing levels of serum alpha-fetoprotein to death in hepatocellular carcinoma is discussed in detail.     

Occurrence of two distinct succinate thiokinases in animal tissues

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.     

Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115]

Recent studies using bee and wasp venom peptides have led to the hypothesis that proper complex formation with calmodulin (CaM) requires the presence of a basic amphiphilic helix on the surface of the target protein [Cox, J. A. (1984) Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 3000]. We have tested this hypothesis by examining CaM and troponin C (TnC) complex formation with two basic peptides, the wasp venom tetradecapeptide mastoparan and the physiologically relevant synthetic troponin I (TnI) inhibitory peptide [104-115], using far-ultraviolet circular dichroism as a secondary structure probe. Complex formation between mastoparan and either CaM or TnC results in an increase in helical content, whereas the helical content of TnI inhibitory peptide does not increase when bound to either protein. Significantly, mastoparan is 78% alpha-helical in a 50% solution of the helix-inducing solvent trifluoroethanol and has a high helix-forming potential according to the Chou-Fasman rules while TnI inhibitory peptide contains none and is not predicted to have any. We interpret these data as indicating that these peptides exhibit substantially different secondary structures upon binding to CaM or TnC. The ability of mastoparan to regulate the acto-subfragment 1-tropomyosin ATPase has also been examined. Mastoparan and TnI inhibitory peptide inhibited 31% and 45% of the activity, respectively. TnC and CaM promote differing degrees of Ca2+-sensitive release of inhibition by both peptides. Sequence comparison suggests that the basic residues present in both peptides are important for binding. However, we conclude that an alpha-helical structure is not a prerequisite for the binding of target proteins to CaM and TnC.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Comparison of material properties in fascicle-bone units from human patellar tendon and knee ligaments

The fascicle material properties in bone-fascicle-bone units were determined for the anterior and posterior cruciate ligaments (ACL, PCL), the lateral collateral ligament (LCL) and the patellar tendon (PT) from three young human donor knees. Groups of fascicles from each tissue were isolated with intact bone ends and failed at a high strain rate in a saline bath at 37 degrees C. In each knee tested the load related material properties (linear modulus, maximum stress and energy density to maximum stress) for the patellar tendon were significantly larger than corresponding values for the cruciate and collateral ligaments. Bundles from different ligaments in the same knee were similar to each other in their mechanical behavior. In addition, no significant differences were present in the maximum strains recorded for any of the four tissue types examined. The results presented have implications in studies of ligament injury. They are also important in the design and use of synthetic and biological ligament replacements and in tissue and whole knee modeling.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Growth of epizootic hemorrhagic disease, akabane, and ephemeral fever viruses in Aedes albopictus cells maintained at various temperatures

The growth curves of one epizootic hemorrhagic disease (EHD) virus serotype (Reoviridae), two Akabane virus strains (Bunyaviridae) and three bovine ephemeral fever (BEF) group viruses (Rhabdoviridae) were determined in Aedes albopictus cells maintained at 15, 20, 28 and 33 degrees C. Ae albopictus cells supported the growth of all the viruses although not necessarily at all temperatures. Because none of the viruses exhibited cytopathic effect in Ae albopictus cells, growth was assayed in baby hamster kidney 21 (BHK21) cells maintained at 37 degrees C. The temperature at which the Ae albopictus cells were maintained had a marked effect on the growth and yield for each virus studied. EHD virus was heat-stable and grew after 4 days at 28 and 33 degrees C, and after 8 days at 20 degrees C. No growth was recorded up to 12 days at 15 degrees C. The two Akabane viruses were heat-sensitive and exhibited different growth patterns. One strain (B8935) showed no growth at 15 degrees C and only minimal growth at 20, 28 and 33 degrees C. The other strain (CSIRO 16) showed growth after 1-2 days at all temperatures with higher titres reached at 15 and 20 degrees C than at 28 and 33 degrees C. The BEF group viruses grew to approximately the same titres at all temperatures. At the higher temperatures (28 and 33 degrees C) most of BEF group viruses had disappeared within 9 days. In contrast at the lower temperatures (15 and 20 degrees C), there was still virus present 18 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple causes of pregnancy failure in hamsters precociously ovulated by human chorionic gonadotropin

Cycling adult female hamsters can be induced to mate and ovulate 24 h early by the injection of 20 IU human chorionic gonadotropin (hCG) at 1500 h on Day 3 (day before proestrus), but pregnancy is not established. Although there is evidence of decreased sperm transport in precociously ovulated females, this does not appear to be the primary cause of infertility. Reduced size and vascularity of corpora lutea (CL) in treated females suggests incomplete or failed CL activation. Control and hCG-treated females were killed by exsanguination under ether anesthesia at intervals for the first 5 days after mating. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, estradiol, and progesterone were measured by radioimmunoassay. Luteinizing hormone in treated animals was very high at 2200 h on Day 1 after mating (31 h after the hCG injection), due to endogenous release, and dropped below control levels thereafter. Follicle-stimulating hormone, by contrast, was significantly lower than controls at 2200 h on Day 1 and remained low until 2200 h on Day 3 after mating. Prolactin in treated animals was not different from that in controls, except for 1000 h on Day 4, when it showed a significant dip. Estradiol in treated animals was significantly higher than in controls at 2200 h on Day 1 (when LH was also high and FSH was low), and remained high at 1000 h and 2200 h on Day 2, dropping thereafter to control levels. Progesterone was initially at control levels but had dropped significantly by 1000 h on Day 2 and remained low for the next 24 h. These results suggest that pregnancy failure is due to inadequate activation of corpora lutea. This may be due to: 1) immaturity of follicles at the time of ovulation; 2) inappropriate timing of preovulatory events; 3) the luteolytic effects of high levels of LH or estradiol or both; 4) the low level of FSH in the early stages of corpus luteum development; or 5) a combination of the above. Abnormalities of prolactin secretion were not investigated in detail but cannot be ruled out at this time.     

Blockade of a motor response by cholinergic stimulation of the toad midbrain tegmentum

Application of an acid solution to the dorsal skin of conscious toads having intact nervous system induces a scratching reflex and escape movements, as well as autonomic alterations (hypertension and tachycardia) that are part of the defense response. The motor components of this response are abolished or reduced by microinjection of 60, 30, 15 or 7.5 ng carbachol into the midbrain tegmentum. The cardiovascular components, however, continue to be present, although their amplitude is reduced. The depression of the motor response is statistically significant up to 15 minutes for the 60 ng dose, up to 10 minutes for the 15 and 30 ng doses, and only up to 5 minutes for the 7.5 ng dose. The data suggest that the midbrain tegmentum may modulate the reflex motor response triggered by a noxious stimulus and also participate in the organization of the escape movements. The importance of cholinergic agents in this modulation is discussed. The persistence of the cardiovascular component of the response shows the importance of this parameter as an indicator of alert situations.