Crystal structure of the alpha-actinin rod reveals an extensive torsional twist

BACKGROUND: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins. RESULTS: We report here the crystal structure of the alpha-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface. CONCLUSIONS: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.     

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.     

Exertion-induced fatigue and thermoregulation in the cold

Cold exposure facilitates body heat loss which can reduce body temperature, unless mitigated by enhanced heat conservation or increased heat production. When behavioral strategies inadequately defend body temperature, vasomotor and thermogenic responses are elicited, both of which are modulated if not mediated by sympathetic nervous activation. Both exercise and shivering increase metabolic heat production which helps offset body heat losses in the cold. However, exercise also increases peripheral blood flow, in turn facilitating heat loss, an effect that can persist for some time after exercise ceases. Whether exercise alleviates or exacerbates heat debt during cold exposure depends on the heat transfer coefficient of the environment, mode of activity and exercise intensity. Prolonged exhaustive exercise leading to energy substrate depletion could compromise maintenance of thermal balance in the cold simply by precluding continuation of further exercise and the associated thermogenesis. Hypoglycemia impairs shivering, but this appears to be centrally mediated, rather than a limitation to peripheral energy metabolism. Research is equivocal regarding the importance of muscle glycogen depletion in explaining shivering impairments. Recent research suggests that when acute exercise leads to fatigue without depleting energy stores, vasoconstrictor responses to cold are impaired, thus body heat conservation becomes degraded. Fatigue that was induced by chronic overexertion sustained over many weeks, appeared to delay the onset of shivering until body temperature fell lower than when subjects were rested, as well as impair vasoconstrictor responses. When heavy physical activity is coupled with underfeeding for prolonged periods, the resulting negative energy balance leads to loss of body mass, and the corresponding reduction in tissue insulation, in turn, compromises thermal balance by facilitating conductive transfer of body heat from core to shell. The possibility that impairments in thermoregulatory responses to cold associated with exertional fatigue are mediated by blunted sympathetic nervous responsiveness to cold is suggested by some experimental observations and merits further study.     

Effects of androgen deprivation on the histology of adult chimpanzee testis

Until primate sperm are exposed to the unique microenvironment of the epididymis, they are not capable of fertilization or vigorous motility. Many of the proteins that contribute to the unique microenvironment of the primate epididymis, and thus to sperm maturation, are dependent on androgens to induce their synthesis and secretion. GnRH antagonists have proved effective in suppressing LH and testosterone synthesis and secretion, and thus in maintaining a state of androgen deprivation or functional hypogonadotropism. We report here the effects of GnRH antagonist-induced androgen-deprivation on the histology of the testicular interstitium and seminiferous epithelium of the adult male chimpanzee. After only 21 days of androgen-deprivation, chimpanzee testicular tissues exhibit specific atrophic changes, including the loss of contact between developing spermatocytes and between Sertoli cells and their developing spermatids, alterations in cell development resulting in missing maturation steps (elongating Sc and structurally complete Sd2 spermatids) and inappropriate cell associations, varying degrees of cytoplasmic degradation in germ cells, Sertoli cells, and Leydig cells, and a tubular lumen obscured by masses of sloughed primary and secondary spermatocytes and what appear histologically to be Sb1 and Sd1 spermatids.     

Intracellular calcium gradients in cultured human uterine smooth muscle: a functionally important subplasmalemmal space

The plasma membrane contains the key elements for the control of coupling excitation to contraction in smooth muscle. The superficial calcium buffer barrier, initially proposed by van Breemen for vascular smooth muscle, may participate in the regulation of calcium entry in other smooth muscle types. To investigate the relationship between the sarcoplasmic reticulum (SR) and the plasma membrane in myometrial smooth muscle cells, we performed experiments using videofluorescence imaging and cell-attached electrophysiology. The cell-attached patch was used as a reporter for the free calcium in the subplasmalemmal space by monitoring openings of the Maxi-K channel. Calcium green-1 was used to simultaneously monitor changes of the deep cytosolic calcium concentrations. The cell with the patch attached was stimulated via an intercellular calcium wave from an adjacent cell. In this fashion, release of SR calcium was accomplished with minimal disturbance of the plasma membrane and the subplasmalemmal space of the cell studied. With physiological bathing solution, six of seven calcium waves activated Maxi-K channels. Surprisingly, the Maxi-K channels began opening 6.3 +/- 4.7s (range 2.6-15.0s) after the wave passed the pipette location. When plasma membrane calcium fluxes were inhibited with 100 microM lanthanum, no Maxi-K channel openings were observed in six of seven experiments. These results are best explained by a subplasmalemmal space in which the calcium concentration is largely controlled by store-operated channels. These results suggest the superficial buffer barrier as merely one aspect of subplasmalemmal regulation of calcium dynamics, and emphasize the importance of store-operated calcium channels during dynamic calcium changes.     

Rapid and simple purification of a novel extracellular β-amylase from Bacillus sp.

Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M _r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-.     

Automated identification of Fos expression

The concentration of Fos, a protein encoded by the immediate-early gene c-fos, provides a measure of synaptic activity that may not parallel the electrical activity of neurons. Such a measure is important for the difficult problem of identifying dynamic properties of neuronal circuitries activated by a variety of stimuli and behaviours. We employ two-stage statistical pattern recognition to identify cellular nuclei that express Fos in two-dimensional sections of rat forebrain after administration of antipsychotic drugs. In stage one, we distinguish dark-stained candidate nuclei from image background by a thresholding algorithm and record size and shape measurements of these objects. In stage two, we compare performance of linear and quadratic discriminants, nearest-neighbour and artificial neural network classifiers that employ functions of these measurements to label candidate objects as either Fos nuclei, two touching Fos nuclei or irrelevant background material. New images of neighbouring brain tissue serve as test sets to assess generalizability of the best derived classification rule, as determined by lowest cross-validation misclassification rate. Three experts, two internal and one external, compare manual and automated results for accuracy assessment. Analyses of a subset of images on two separate occasions provide quantitative measures of inter- and intra-expert consistency. We conclude that our automated procedure yields results that compare favourably with those of the experts and thus has potential to remove much of the tedium, subjectivity and irreproducibility of current Fos identification methods in digital microscopy.     

Time zones: a comparative genetics of circadian clocks

The circadian clock is a widespread cellular mechanism that underlies diverse rhythmic functions in organisms from bacteria and fungi, to plants and animals. Intense genetic analysis during recent years has uncovered many of the components and molecular mechanisms comprising these clocks. Although autoregulatory genetic networks are a consistent feature in the design of all clocks, the weight of evidence favours their independent evolutionary origins in different kingdoms.