Cell cycle effects of an autologous growth promoter from human leukemia cells

The mechanism of action of an autologous growth promoter, produced by HL-60 cells grown in serum-free defined medium, was investigated. Determinations of cell cycle phase distribution, which showed an approx. 16% increase in S and G2M with a reciprocal decrease in G1 cells, indicated a mechanism causing decreased time in G1, resulting in a more rapid entry of cells into G2M. Adsorption of the culture supernatant with HL-60 cells resulted in a decreased promoter activity, suggesting an autologous receptor mechanism for the promoter.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits.

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.     

Quantification of cuttlefish (Sepia officinalis) camouflage: a study of color and luminance using in situ spectrometry

Cephalopods are renowned for their ability to adaptively camouflage on diverse backgrounds. Sepia officinalis camouflage body patterns have been characterized spectrally in the laboratory but not in the field due to the challenges of dynamic natural light fields and the difficulty of using spectrophotometric instruments underwater. To assess cuttlefish color match in their natural habitats, we studied the spectral properties of S. officinalis and their backgrounds on the Aegean coast of Turkey using point-by-point in situ spectrometry. Fifteen spectrometry datasets were collected from seven cuttlefish; radiance spectra from animal body components and surrounding substrates were measured at depths shallower than 5 m. We quantified luminance and color contrast of cuttlefish components and background substrates in the eyes of hypothetical di- and trichromatic fish predators. Additionally, we converted radiance spectra to sRGB color space to simulate their in situ appearance to a human observer. Within the range of natural colors at our study site, cuttlefish closely matched the substrate spectra in a variety of body patterns. Theoretical calculations showed that this effect might be more pronounced at greater depths. We also showed that a non-biological method (“Spectral Angle Mapper”), commonly used for spectral shape similarity assessment in the field of remote sensing, shows moderate correlation to biological measures of color contrast. This performance is comparable to that of a traditional measure of spectral shape similarity, hue and chroma. This study is among the first to quantify color matching of camouflaged cuttlefish in the wild.     

Cx36 expression in the AII‐mediated rod pathway is activity dependent in the developing rabbit retina

Gap junctions are composed of connexin 36 (Cx36) and play a critical role in the rod photoreceptor signaling pathways of the vertebrate retina. Despite the fact that their connection and modulation in various rod pathways have been extensively studied in adult animals, little is known about the contribution and regulation of gap junctions to the development of the AII amacrine cell (AC)‐mediated rod pathway. Using immunohistochemistry and microinjection, this study demonstrates a steady increase in relative Cx36 protein expression in both plexiform layers of the rabbit retina at around the time of eye opening. However, immediately after eye opening, most Cx36 immunoreactive AII ACs show no gap junction coupling pattern to neighboring cells and it is not until the third postnatal week that AII cells begin to exhibit an adult‐like tracer‐coupling pattern. Moreover, studies using dark‐rearing and AMPA receptor blockade during postnatal development both revealed that relative levels of Cx36 immunoreactivity in AII ACs were increased when neural activity was inhibited . Our findings suggest that Cx36 expression in the AII‐mediated rod pathway is activity dependent in the developing rabbit retina . © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 473–486, 2016