Electron microscopic and electron radioautographic study of cells in glandular hyperplasia and cancer of the endometrium

Peculiarities of the ultrastructure and RNA synthesis in the epithelial endometrial cells of nine patients with hyperplasia and the uterine cavity cancer have been studied by the electron-radioautographic method. Quantitative differences are revealed in the ultrastructures of the cells. Amplification of the nucleoli labelling and asynchronism of the RNA synthesis in the nucleoli with glandular hyperplasia and endometrial cancer are observed.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. IX. The effect of the pH and ionic strength of the solution on the conformational restructurings of F-actin induced by the binding of heavy meromyosin

The dependence of F-actin conformational changes induced by the F-actin-HMM complex on pH and ionic strength was found by polarized ultraviolet fluorescence microscopy. It is discovered that pH affects sufficiently the cooperativity of F-actin structural changes, while the ionic strength affects their depth. The actomyosin complex was supposed to be at least in two structural states, differing in their orientation as well as in flexibility of F-actin monomers.     

Sensitivity to stress in the bivalve Macoma balthica from the most northern (Arctic) to the most southern (French) populations: low sensitivity in Arctic populations because of genetic adaptations?

The stress sensitivity, determined in copper exposureexperiments and in survival in air tests, and thegenetic structure, measured by means of isoenzymeelectrophoresis, were assessed in populations of theBaltic clam Macoma balthica (L.) from itssouthern to its northern distribution limit, in orderto test the hypotheses that near the distributionlimit the clams would be more stress sensitive andwould have a lower genetic variability. Thepopulations in west and north Europe show a stronggenetic resemblance. The populations in the sub-ArcticWhite Sea are genetically slightly different, and showa low stress sensitivity. The populations in theArctic Pechora Sea are genetically very distant fromthe other populations, and show the lowest stresssensitivity. Near the southern distribution limit, inagreement with the hypotheses, genetic variability islow and stress sensitivity high. On the other hand, incontrast to expectation, near the northerndistribution limit, in the populations of the PechoraSea, the genetic variability was higher, thus notreduced, and the stress sensitivity was low comparedto all other populations. Yet, it remains a questionif such is due to gradual physiologicalacclimatization (and ongoing differential selection)or to genetic adaptation.     

Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD⁺ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD⁺-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD⁺ triggered Golgi disassembly by BFA. This effect of NAD⁺ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

The cole-moore effect in nodal membrane of the frogRana ridibunda: Evidence for fast and slow potassium channels

Summary The K conductance (g _K) kinetics were studied in voltage-clamped frog nodes (Rana ridibunda) in double-pulse experiments. The Cole-Moore translation forg _K–t curves associated with different initial potentials (E) was only observed with a small percentage of fibers. The absence of the translation was found to be caused by the involvement of an additional, slow,g _K component. This component cannot be attributed to a multiple-state performance of the K channel. It can only be accounted for by a separate, slow K channel, the fast channel being the same as then ⁴ K channel inR. pipiens.The slow K channel is characterized by weaker sensitivity to TEA, smaller density, weaker potential (E) dependence, and somewhat more negativeE range of activation than the fast K channel. According to characteristics of the slow K system, three types of fibers were found. In Type I fibers (most numerous) the slow K channel behaves as ann ⁴ HH channel. In Type II fibers (the second largest group found) the slow K channel obeys the HH kinetics within a certainE range only; beyond this range the exponential decline of the slowg _K component is preceded by anE-dependent delay, its kinetics after the delay being the same as those in Type I fibers. In Type III fibers (rare) the slow K channel is lacking, and it is only in these fibers that the Cole-Moore translation of the measuredg _K–t curves can be observed directly.The physiological role of the fast and slow K channel in amphibian nerves is briefly discussed.     

Base-Calling Algorithm with Vocabulary (BCV) Method for Analyzing Population Sequencing Chromatograms

Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3–14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.