Thin filaments are not of uniform length in rat skeletal muscle.

The variation in thin filament length was investigated in slow and fast muscle from adult and neonatal rats. Soleus (slow) muscle from adult, 3-, 7-, and 9-d-old rats, and extensor digitorum longus (EDL; fast) muscle from adult rats were serially cross-sectioned. The number of thin filaments per 0.06 microns2 (TF#) was counted for individual myofibrils followed from the H zone of one sarcomere, through the I-Z-I region, to the H zone of an adjacent sarcomere TF# was pooled by distance from the Z band or AI junction. In both adult muscles, thin filament length varied from 0.18 to 1.20 microns, with approximately 25% of the thin filaments less than 0.7 microns in length. In 7- and 9-d soleus, thin filament length ranged from 0.18 to 1.08 microns; except for the longest (0.18 to 1.20 microns) filaments, the distribution of thin filament lengths was similar to that in adult muscle. In 3-d soleus, thin filament length was more uniform, with less than 5% of the filaments shorter than 0.7 microns. In all neonatal muscles, there were approximately 15% fewer thin filaments per unit area as compared to adult muscles. We conclude: (a) In rat skeletal muscle, thin filaments are not of uniform length, ranging in length from 0.18 to 1.20 microns. (b) There may be two stages of thin filament assembly in neonatal muscle: between 3 and 7 d when short thin filaments may be preferentially or synthesized or inserted near the Z-band, and between 9 d and adult when thin filaments of all lengths may be synthesized or inserted into the myofibril.

Microtubules in mammalian heart muscle.

Sections of adult mammalian cardiac muscles fixed at room temperature reveal numerous microtubules (24--28 nm in diameter) both near the nucleus and in the extra-myofibrillar space. Microtubules encircle the nucleus, are associated with the myofibrils in a helical arrangement, and form a network that runs transversely at the level of the I band and axially between the myofibrils. Microtubules are more numerous in muscle cells than previously recognized and may perform more than cytoskeletal function.

Optical diffraction of the Z lattice in canine cardiac muscle.

Optical diffraction patterns from electron micrographs of both longitudinal and cross sections of normal and anomalous canine cardiac Z bands have been compared. The data indicate that anomalous cardiac Z bands resembling nemaline rods are structurally related to Z bands in showing a repeating lattice common to both. In thin sections transverse to the myofibril axis, both electron micrographs and optical diffraction patterns of the Z structure reveal a square lattice of 24 nm. This lattice is simple at the edge of each I band and centered in the interior of the Z band, where two distinct lattice forms have been observed. In longitudinal sections, oblique filaments visible in the electron micrographs correspond to a 38-nm axial periodicity in diffraction patterns of both Z band and Z rod. We conclude that the Z rods will be useful for further analysis and reconstruction of the Z lattice by optical diffraction techniques.

The Z lattice in canine cardiac muscle.

Filtered images of mammalian cardiac Z bands were reconstructed from optical diffraction patterns from electron micrographs. Reconstructed images from longitudinal sections show connecting filaments at each 38-nm axial repeat in an array consistent with cross-sectional data. Some reconstructed images from cross sections indicate two distinctly different optical diffraction patterns, one for each of two lattice forms (basket weave and small square). Other images are more complex and exhibit composite diffraction patterns. Thus, the two lattice forms co-exist, interconvert, or represent two different aspects of the same details within the lattice. Two three-dimensional models of the Z lattice are presented. Both include the following features: a double array of axial filaments spaced at 24 nm, successive layers of tetragonally arrayed connecting filaments, projected fourfold symmetry in cross section, and layers of connecting filaments spaced at intervals of 38 nm along the myofibril axis. Projected views of the models are compared to electron micrographs and optically reconstructed images of the Z lattice in successively thicker cross sections. The entire Z band is rarely a uniform lattice regardless of plane of section or section thickness. Optical reconstructions strongly suggest two types of variation in the lattice substructure: (a) in the arrangement of connecting filaments, and (b) in the arrangement of units added side-to-side to make larger myofilament bundles and/or end-to-end to make wider Z bands. We conclude that the regular arrangement of axial and connecting filaments generates a dynamic Z lattice.

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle.

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three-dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z-filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.

DNA synthesis and mitosis in well-differentiated mammalian cardiocytes.

Incorporation of [(3)H]thymidine into nuclei of heart cells of 2-day-old rats indicates that neonatal cardiac cells containing well-aligned myofibrils synthesize DNA. In these highly differentiated cells, neither the presence of contractile proteins nor their organization into myofibrils inhibits either DNA synthesis or mitosis.

Sarcoplasmic reticulum of striated muscle: localization of potential calcium binding sites.

Fish branchial muscle stained at a low pH with thorium dioxide shows localization of the stain over the sacroplasmic reticulum. Binding of the positively charged thorium micelles with dissociated acid groups of polyanions in this region suggests a possible mechanism for the storage and release of divalent cations such as calcium.

Calcium uptake by two preparations of mitochondria from heart.

Ca/+ transport and respiratory characteristics of two preparations of cardiac mitochondria (Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) J. Biol. Chem. 252, 8731-8739) isolated using polytron homogenization (subsarcolemmal mitochondria) and limited Nagarse exposure (intermyofibrillar mitochondria) are described. The Nagarse procedure yields mitochondria with 50% higher rates of oxidative phosphorylation than the polytron-prepared mitochondria in both rat and dog. Rat hear intermyofibrillar mitochondria contain 50% more cytochrome aa3 than the polytron preparation, whereas in the dog, cytochrome aa3 content is not significantly different. Cytochrome oxidase activities and cytochrome c, c1 and b contents were comparable in both populations of rat and dog heart mitochondria. The V of succinate-supported Ca2+ accumulation for Nagarse-prepared mitochondria from rat heart was 1.8-fold higher than the polytron-prepared mitochondria. In dog heart, the Nagarse preparation showed a 3.0-fold higher V for Ca2+ uptake compared to the polytron preparation. A lower apparent affinity for Ca2+ was demonstrated in the intermyofibrillar mitochondria for both species (Km is 2-2.5-fold higher). The Hill coefficient was 1 both mitochondrial types. Subsarcolemmal mitochondria from both species were treated with Nagarse to determine the role of this treatment on the observed differences. Nagarse did not alter any kinetic parameter of Ca2+ uptake. The properties of these mitochondria with reference to their presumed intracellular location may pertain to the role of mitochondria as an intracellular Ca2+ buffering mechanism in contractile tissue.

Structural states in the Z band of skeletal muscle correlate with states of active and passive tension.

In skeletal muscle Z bands, the ends of the thin contractile filaments interdigitate in a tetragonal array of axial filaments held together by periodically cross-connecting Z filaments. Changes in these two sets of filaments are responsible for two distinct structural states observed in cross section, the small-square and basketweave forms. We have examined Z bands and A bands in relaxed, tetanized, stretched, and stretched and tetanized rat soleus muscles by electron microscopy and optical diffraction. In relaxed muscle, the A-band spacing decreases with increasing load and sarcomere length, but the Z lattice remains in the small-square form and the Z spacing changes only slightly. In tetanized muscle at sarcomere lengths up to 2.7 micron, the Z lattice assumes the basketweave form and the Z spacing is increased. The increased Z spacing is not the result of sarcomere shortening. Further, passive tension is not sufficient to cause this change in the Z lattice; active tension is necessary.

Physiological, biochemical, and morphological characteristics of myocardial anoxia: the use of a semi-perfusion canine preparation.

Regional myocardial anoxia was produced in dogs by perfusion of the left circumflex artery (LCA) with deoxygenated blood. Isolated sarcoplasmic reticulum fragments (cardiac relaxing system) showed decreased Ca2+ binding and uptake. The ability of isolated mitochondria to utilise long-chain fatty acids was markedly reduced. This model has revealed inherent biochemical differences between ischaemia and anoxia.

Targeted developmental overexpression of calmodulin induces proliferative and hypertrophic growth of cardiomyocytes in transgenic mice.

Calmodulin (CaM) levels are developmentally regulated in the mouse heart. During late gestational and early postnatal stages, CaM levels decline several-fold in close temporal association with the declining population of proliferating cardiomyocytes. This correlation suggests that CaM may influence cardiomyocyte cell cycle activity, particularly since CaM is implicated in cell cycle control in several eukaryotic nonmuscle cells. To test this possibility, nucleotides -500 to 77 of the human atrial natriuretic factor gene were linked to a chicken CaM minigene to establish two pedigrees of transgenic mice that express 3- to 5-fold increased levels of CaM in cardiomyocytes. Developmental overexpression of CaM in mouse cardiomyocytes produced a markedly exaggerated cardiac growth response, characterized by the presence of cardiomyocyte hypertrophy in regions demonstrated to overexpress CaM and by cardiomyocyte hyperplasia, apparent at early developmental stages. Early postnatal suppression of fusion gene expression in the cardiac ventricles correlated with regression of the ventricular growth response in transgenic relative to nontransgenic mice between 3 days and 6-10 weeks of age, but was not apparent in the cardiac atria, where levels of CaM remained constitutively elevated until advanced stages. To test the possibility that increased cytosolic Ca2+ buffering contributes to the growth response induced by CaM over-expression, two additional lines of transgenic mice were generated using the same human atrial natriuretic factor promoter to target expression of a CaM mutant (amino acids 75-82 deleted) in cardiomyocytes. This mutant has previously been shown to bind Ca2+ with kinetic properties similar to those of wild-type CaM, but was unable to activate several CaM-dependent target enzymes in vitro. Despite high level expression of the CaM mutant, no growth response was apparent in the hearts of transgenic relative to those of nontransgenic mice, suggesting that increased Ca2+ buffering is unlikely to contribute to the growth response induced by CaM overexpression. Taken together, these findings reveal that cardiomyocyte growth regulation is specifically influenced by CaM concentrations in transgenic mice.

Prokaryotic promoters in biotechnology.

The basic properties of prokaryotic promoters and the promotor region are described with special emphasis on promoters that are found in Escherichia coli and Bacillus subtilis. Promoters recognized by major and minor forms of RNA polymerase holoenzymes are compared for their specificities and differences. Both natural and hybrid promoters that have been constructed for purposes of efficient and regulated transcription are discussed in terms of their utility. Since promoter regions contain sequences that are recognized not only by RNA polymerase but by positive and negative regulatory factors that regulate expression from promoters, the functions and properties of these promoter regions are also described. The current utility and the future prospects of the prokaryotic promoters in expressing heterologous genes for biotechnology purposes are discussed.

Cardiac morphology after conditions of microgravity during COSMOS 2044.

Light- and electron-microscopic studies were performed on cardiac muscle from rats flown on COS-MOS 2044 and from four control groups. Average cross-sectional area of myofibers was measured by video analysis of the light-microscopic images of papillary and ventricular muscle samples from all animals. This cross-sectional area was significantly decreased in flight rats (P = 0.03) compared with synchronous controls. Additional findings at the electron-microscopic level consistent with this atrophy were obtained by stereological analysis and optical diffraction analysis of papillary muscle samples. Slightly higher mitochondrial volume density values and mitochondria-to-myofibril ratios as well as normal A-band spacings (d1,0) and Z-band spacings of myofibrils were observed in the tail-suspension and flight groups. General morphological features similar to those in ventricular samples from the previous COSMOS 1887 flight were observed.

Alterations in cell free brain protein synthesis following ethanol withdrawal in physically dependent rats.

The effects of ethanol withdrawal were determined on cell free brain protein synthesis in physically dependent rats. Following the development of physical dependence, ethanol abstinence for 24 h resulted in decreased protein synthesis in cerebral tissue. The observed inhibition of [14C]leucine incorporation into protein was found to be reversible after 7 days of ethanol withdrawal. Although the ribosomes from control, ethanol-treated and ethanol-withdrawn animals were highly responsive to polyuridylic acid stimulation, the ribosomes from the control group consistently exhibited higher activity. The determination of protein content of the ribosomal fraction showed a significant increase following ethanol administration and was further enhanced by ethanol abstinence. The results suggest that ethanol-induced changes at the ribosomal level may result in defective association of mRNA causing depression of brain protein synthesis.

Superoxide generation by photomediated redox cycling of anthraquinones.

Anthraquinones (AQs) comprise one important class of secondary metabolites predominantly produced by fungi and higher plants but also produced by a variety of other organisms. Humans orally ingest AQs from environmental sources as well as through direct use as nonprescription laxatives, and some AQ derivatives are used as topically applied antipsoritic agents. Some AQs are mutagenic. We present evidence that aqueous solutions of several AQs in the presence of an appropriate reducing agent and dissolved oxygen generate superoxide when they are illuminated with broad-spectrum light. Redox cycling of AQs could be responsible for some aspects of their toxicity in biological systems.

Perinuclear microtubules in postnatal rat heart.

Cytoplasmic microtubules can be divided into two subpopulations: 1) those adjacent to the nucleus (perinuclear), and 2) those distributed between the myofilament bundles (nonperinuclear). Previous observations (Cartwright and Goldstein, '83) indicate total cytoplasmic microtubule numeric density increases to a maximum at 5-9 days and decreases to the steady value of the adult muscle. We have examined the numeric density (mean numbers of microtubule profiles per micron2 cross-sectional area) of the perinuclear subpopulation and compared it to the numeric density of the total cytoplasmic microtubule population in postnatally developing rat papillary muscle ages 1, 3, 5, 9, 21, and 42 days, and adult. The perinuclear region was defined as the area around the nucleus which extends to the 0.273 micron from the nuclear envelope. The density of perinuclear microtubules did not change with postnatal development. Our study suggests that perinuclear microtubules are a separate and relatively stable subpopulation of the total population of cytoplasmic microtubules and may serve a function different from that of the more variable nonperinuclear microtubules.

Hepatitis B immunization in adolescents.

This article reviews the epidemiology of hepatitis B in the United States, previous vaccination strategy, and reasons for its failure and issues leading to the recommendation to vaccinate all adolescents. A review of specific hepatitis B virus risk behaviors of adolescents and barriers to vaccinating adolescents is covered. Strategies that favor successful completion of the immunization series are also examined. Hepatitis B infection is an important public health concern for adolescents. The previous vaccine strategy to immunize only individuals though to be at high risk was unsuccessful, especially because providers of care could not identify these individuals. Furthermore, many individuals thought not to be at high risk for infection were exposed through contacts which could not be identified. Challenges to immunization of adolescents include logistical issues, patient education, cost of the vaccine, and patient compliance. Several of these issues can be addressed by a school-based hepatitis B immunization program. The body of evidence and national policy is rapidly changing to support the recommendation that all adolescents receive the hepatitis B immunization series. The series would be most effective if administered during the middle-school years. A universal adolescent hepatitis B vaccination program would result in the most immediate health benefits and acceleration toward the eradication of hepatitis B in the United States.

The effects of increased gravity and microgravity on cardiac morphology.

BACKGROUND: Our previous study of rats exposed for 14 d to microgravity on Cosmos 2044 revealed morphological changes consistent with cardiac atrophy. METHODS: In the current comparison study, light and electron microscopic studies were performed on cardiac muscle from 10 rats exposed to hypergravity (continuous centrifugation at 2G) for 14 d. RESULTS: Myofiber area was significantly greater in the 2G papillary muscle as compared with muscle from 10 control rats of the same strain and size. This contrasts with the significant decrease in myofiber area previously seen in the rats exposed to microgravity. At the electron microscopic level, general morphological features were similar in both groups and resembled tissue from control rats from the previous Cosmos studies. However, mitochondria from papillary and ventricular muscle from the 2G rats revealed signs of fatigue typical of the early stage of hypertrophy. These results are consistent with a state of adaptive cardiac hypertrophy for the 2G group.

Gaze as a function of conversation and degree of love.

The possibility that Rubin's findings of greater mutual gazing between strong than weak lovers and between strangers could be attributed to the occurrence of more conversation in strong love conditions was tested. Ss were selected from an undergraduate population. Ten strong love couples, determined through the use of Rubin's love scale, were compared to 10 pairs of unacquainted Ss for the amount of mutual eye contact, as well as conversation time and time spent in pure gazing without conversation. It was found that lovers did converse more with each other than with strangers. However, they also spent more time in pure gazing during periods of silence, lending credence to Rubin's results. The methodological implications of using videotape for eye contact studies were also discussed.