Cell differentiation and pigment composition in Anabaena cylindrica

Summary Cell differentiation in Anabaena cylindrica is accompanied with characteristic changes in the pigment composition of heterocysts and spores. In both the absence of phycocyanin is consistent with the lack of CO₂-fixing ability previously reported. The presence of chlorophyll and -carotene suggests a functional photosystem I in heterocysts. In the spores chlorophyll is largely replaced by pheophytin. The quantitative distribution of carotenoids is also affected. An increase in the proportion of -carotene is characteristic of heterocysts while spores show a larger proportion of xanthophylls compared with the intact filament.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intersubunit fluorescence energy transfer in human factor VIII

Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.     

Periodic organization of the contractile apparatus in smooth muscle revealed by the motion of dense bodies in single cells

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns of motion. Analysis of the relative motion of the bodies indicated that some bodies were structurally linked to one another or constrained so that the distance between them remained relatively constant during contraction. Such bodies tended to fall into laterally oriented, semirigid groups found at approximately 6-microns intervals along the cell axis. Other dense bodies moved rapidly toward one another axially during contraction. Such bodies were often members of separate semirigid groups. This suggests that the semirigid groups of dense bodies in smooth muscle cells may provide a framework for the attachment of the contractile structures to the cytoskeleton and the cell surface and indicates that smooth muscle may be more well-ordered than previously thought. The methods described here for the analysis of the motion of intracellular structures should be directly applicable to the study of motion in other cell types.     

Differential regulation of IL 6, IL 1 A, IL 1 beta and TNF alpha production in LPS-stimulated human monocytes: role of cyclic AMP.

Interleukin 6 (IL 6), IL 1 alpha, IL beta and tumor necrosis factor (TNF) alpha are four cytokines induced in monocytes by lipopolysaccharide (LPS); however, it is unclear whether the mechanisms which control their production are similar. In this study, we report the effects of prostaglandin E2 (PGE2), and two other cAMP-elevating agents, dibutyryl cAMP and 3-isobutyl-1-methyl-xanthine, on the in vitro LPS-induced production of IL 6, IL 1 alpha, IL 1 beta and TNF alpha by human monocytes. The production of these four cytokines was found to be selectively regulated in monocytes, by increases in intracellular cAMP levels. In effect, such agents enhanced, in a dose-dependent manner, both extracellular and cell-associated IL 6 production by LPS-stimulated monocytes. In contrast, it was confirmed, using the same samples, that these cAMP-elevating agents inhibit both extracellular and cell-associated TNF alpha production in a dose-dependent manner. IL 1 alpha and IL 1 beta production, measured by means of specific immunoreactive assays, were not significantly modified. Kinetic analysis showed that the potentiating effect of cAMP on IL 6 production, along with its inhibiting effect on TNF alpha production, could be seen as early as 1 hr after LPS stimulation. These results demonstrate that IL 6, TNF alpha, IL 1 alpha and IL 1 beta production can be differently modulated by an agent, PGE2, which is produced simultaneously by LPS-stimulated monocytes. Such differential autocrine modulation may play an important role in the regulation of the production of cytokines participating in immune and inflammatory responses.     

Acoustic response properties of single units in the torus semicircularis of the goldfish,Carassius auratus

Single units of the goldfish torus semicircularis (TS) were recorded in response to pure tones. Response areas (RA) were obtained by recording the number of spikes evoked by tones in a range of frequencies and levels within the units' dynamic range. RAs gave estimates of best sensitivity (BS), characteristic frequency (CF), most excitatory frequency at each level (BF), and Q_10dB. Peri-stimulus-time histograms (PSTH), interspike interval histograms (ISIH), and period histograms were obtained at various frequencies and levels to describe the units' temporal response patterns.The distribution of CF is nonuniform with modes at 155, 455, and 855 Hz. The distribution of the coefficient of synchronization to standard tones is also nonuniform, revealing a dichotomy between units with little or no phase-locking and those that phase-lock strongly. PSTHs for units without significant phase-locking vary widely and include patterns resembling those of the mammalian auditory brainstem. Compared with saccular afferents, torus units tend to have lower spontaneous rates, greater sensitivity, and sharper tuning. Unlike saccular afferents, BF is independent of level for most torus units. Some torus units are similar to saccular afferents while others reveal significant transformations of information between the periphery and the midbrain.Abbreviations BF best frequency - BS best sensitivity - CF characteristic frequency - ISIH inter-spike interval histogram - PSTH peri-stimulus-time histogram - RA response area - TS torus semicircularis     

Suppression of reproductive development and longevity in the redshouldered leaf beetle,Monolepta australis (Col.: Chrysomelidae) by the tachinid,Monoleptophaga caldwelli (Dipt.)

This paper examines the level and seasonal incidence of parasitism of the redshouldered leaf beetle,Monolepta australis (Jacoby), by the tachinid,Monoleptophaga caldwelli Baranov, and assesses the impact of this fly on the beetle's reproductive development and longevity. Highest average numbers ofM. australis were trapped at light in north Queensland in January and February while parasitism peaked at 6.6 and 6.8% in June and July respectively, averaging 4.8% over the entire sampling period and reaching 28% for a single occasion. Overall, only 2.4% of parasitized beetles were represented in the final two stages of ovarian development compared to 29.1% of non-parasitized ones. There was a highly significant (P<0.001) difference in the distribution frequencies of parasitized and non-parasitized females in the various stages of ovarian development, and most parasitized males possessed atrophied testes. For field-collected beetles maintained in the laboratory, average longevity for those parasitized was 15.6 days compared to 72.2 days for those not parasitized.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.