Effects of shearing on chromatin structure

The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.     

Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

Folding crystallization of DNA. Circular dichroism studies.

CD spectra of DNA monocrystals are extremely different from spectra of psi-DNA or DNA-histone H1 or DNA-polylysine complexes. They are discussed to be dependent on the supramolecular organization of DNA in the condensed form, and they are not in contradiction to the previously proposed seven folding model of DNA in chromatin. Conclusions are drawn with regard to the interpretation of chromatin CD spectra.     

Chromatin phospholipid changes during rat liver development

The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0.47-0.59 per cent of total chromatin in newborn animals and 0.22 per cent in 45-day-old animals. No such age-related differences are observed in the nuclei. The phospholipid composition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine.     

Surgical Treatment of Mitral Insufficiency

Sixty-four patients with pure mitral insufficiency were operated upon. Thirty of them had torn chordae tendineae. It was possible to repair the mitral valve in 57 patients and there were five operative deaths. One patient had a femoral artery embolus and another had a cerebral embolus. The incidence of peripheral embolization was 4 per cent compared with 40 per cent reported for ball valve replacement.Forty-eight of the 57 patients with repair (84 per cent) were living and well with at most a grade II/VI apical systolic murmur up to seven and a half years after operation. There has been no evidence of recurrence in these patients.In approximately 90 per cent of patients with pure mitral insufficiency, repair should be performed. When feasible, repair is more satisfactory than valvular replacement, with not only excellent long-term results, but far less morbidity than is reported with ball valve replacement.     

Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin.

Formaldehyde (HCHO) fixation of chicken erythrocyte chromatin produces a marked decrease in its positive circular dichroism (CD), above 260 nm, and the appearance of s small negative ellipticity around 295 nm. The ultraviolet spectrum of chromatin is unaffected, nor does HCHO produce any changes in the uv or CD spectra of chicken erythrocyte DNA. The extent of the circular dichroism transition from the native chromatin to the suppressed spectrum is dependent on the concentration of HCHO and salt concentration. The kinetics of the reactions are complex, implicating at least two reactive species. Studies of the reaction of HCHO with chromatin in ethylene glycol and CD measurements of aqueous chromatin solution with added glutaraldehyde preclude simple dehydration and general cross-linking effects as causes of the CD changes observed. The results are interpreted as indicating a conformational change of the DNA in chromatin caused by histone-DNA or histone-histone cross-linking.     

Time of replication of genes responsible for a temperature-sensitive function in a cell cycle-specific ts mutant from a hamster cell line

A method involving short pulses of 5-bromodeoxyuridine (brUdRib) followed by irraidation with 313 nm light was used to locate the time of replication of certain genes during the cell cycle of two cell lines, AF8 and AL106. AF8, a temperature-sensitive mutant of BHK21/13 cells, grows at 33°C but not at 39.5°C. AL106, a hybrid clone of tsAF8 and SV-40 transformed Lesch-Nyhan fibroblasts (LNSV), which retains all hamster chromosomes and one human chromosome (No. 3), has the ability to grow at 39.5°C. AF8 and AL106 cells synchronized at the G₁-S boundary were released from their block and pulsed with brUdRib for 2-hour periods during the S phase. The cells were subsequently irradiated with 313 nm light. Colony-forming efficiency and revertants frequency were studied. Incorporation of brUdRib during the early S phase (0–4 hours from the begining of S), decreased the colony-forming efficiency of AL106 cells both at 33°C and 39.5°C, and also of AF8 cells at 33°C. No AF8 colonies grew at the nonpermissive temperature regardless of the treatment. Thus the time of replication of genes responsible for colony-forming ability was the same in tsAF8 at the permissive temperature and in AL106 at both temperatures. The time of replication of the genes responsible for the ts function in AF8 cells was located by determining the revertants frequency in synchronized AF8 cells pulsed with brUdRib and irradiated during 1- to 2-hour periods of the S phase. Back-mutants were scored by counting the number of clones capable of growing at 39.5°C (nonpermissive for AF8 cells). The highest frequency of induced back-mutations occurred in synchronized AF8 cells pulsed with brUdRib (and irradiated) between two to four hours from the begining of the S phase. Exposure to brUdRib during other periods of the S phase or during G₁ had no effect on the reversion rate. This method can be used to locate the time of replication (in S) of ts genes in other temperature-sensitive mutants or of other specific genes in other conditional mutants.     

Accessibility of the minor groove of DNA in chromatin to the binding of antibiotics netropsin and distamycin A

The interaction of the antibiotics distamycin A, distamycin analogue and netropsin with chromatin of calf thymus has been studied by circular dichroism measurements and by gel filtration. The minor groove of DNA in chromatin is accessible by 83–89% to the binding of these antibiotics as compared with that of free DNA. The present results combined with our data on the methylation of chromatin with dimethylsulphate [3] strongly suggest that the minor groove of DNA in chromatin is not occupied by chromatin proteins.Abbreviations DM distamycin A - DM₂ analogue of distamycin - Nt netropsin - CD spectra circular dichroism spectra     

The conformation of proteins in chromatin. A circular dichroism study below 250 nm.

This paper is an investigation of the circular dichroism (CD) spectra of DNA and protein in chromatin. The circular dichroism (CD) of chromatin below 250 nm is due to DNA and protein peptide chromophores. The spectrum in this region is resolved into contributions from salt-extractable proteins (histone and non-histone proteins extractable with sodium chloride), residual non-histone proteins (not extractable with 3 M sodium chloride), and DNA. Below 250 nm, DNA in chromatin has the same CD spectrum as DNA free in solution, in contrast to the CD of DNA above 250 nm (Hjelm, R. and Huang, R. C., (1974), Biochemistry 13, 5275). Histones and salt-extractable non-histone proteins in chromatin are seen to have an average CD like those observed for globular proteins. The average CD of the residual non-histone proteins is consistent with a population of proteins with more extended conformation. The CD of each of these components is found to be the same in chromatins isolated from tissues having different nuclear synthetic activities: chick embryo brain, pig cerebellum, myeloma K41, calf thymus, and chicken erythrocyte.     

Binding of progesterone by rat uterus in vitro

Circular dichroism (CD) spectra have been determined for chromatin fractions obtained by ECTHAM-cellulose chromatography. The molecular ellipticity at the positive long wavelength maximum is about 3000 deg cm²/dmol for early-eluted chromatin fractions, thought to be relatively repressed $\text{in vivo}$, and 5000–6000 deg cm²/dmol for late-eluted chromatin fractions, those thought to be preferentially transcribable $\text{in vivo}$. CD bands in the peptide bond spectral region also differ for the two chromatin fractions, early-eluted chromatin having a more helical conformation for proteins. In addition to previously known differences in protein content, the biological activity of a native chromatin fraction can now be correlated with the conformation of its DNA.     

Incorporation of antigen 125I IgG into particular cell compartments: binding by chromatin

Incorporation of [125I]IgG into spleen cells was studied in vivo and in vitro. In vivo, the antigen after uptake into the cytoplasm migrated into cell nuclei, where it was bound to chromatin up to the saturation level. One day after immunization the constant level of [125I]IgG was 1.3 X 10(12) molecules per spleen (10(8) cells). The same number of [125I]IgG molecules were bound to chromatin in cell cultures. The uptake of [125I]IgG was competitively inhibited by non-labelled IgG. Binding of [125I]IgG molecules reextracted from cytoplasm and chromatin with specific anti-human IgG serum argues against the uptake of degraded [125I]IgG molecules. [125I]IgG was tightly bound to DNA. Approximately 50 per cent of [125I]IgG was present in the residual chromatin fraction (after removal of 0.35 M and 2 M NaCl-soluble fractions) and 40 per cent was complexed with DNA (after removal of histones and non-histones AP1, AP2, AP3 and AP4). Binding of [125I]IgG by isolated chromatin was inhibited by the cytoplasmic fraction but not by BSA. Binding of [125I]IgG by fractionated chromatin, (chromatins remaining after removal of 0.35M, and 2M NaCl-soluble fractions or histones + non-histones AP1 + AP2 + AP3 + AP4) occurred at a level similar to that observed with native chromatin. The results suggest that interaction of antigen with immunocompetent cells is not restricted to the cell surface but that antigen seems to be taken up into cytoplasm, migrates to the nuclei and is bound to chromatin, probably directly to DNA. The results are discussed in relation to the induction of the immune reaction.     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

Drug resistance of opportunistic enterobacteria isolated from various sources]

Resistance of 159 strains of opportunistic enterobacteria to 9 antibacterial drugs was studied. The strains were isolated from man and cattle. It was shown that the overwhelming majority of the isolates (93 per cent) were polyresistant irrespective of the genus. There was a high frequency of the strains resistant to the widely used antibiotics such as chloramphenicol (73 per cent), ampicillin (73.6 per cent) and rifampicin (95.6 per cent) and sulfanilamides (99.3 per cent). Gentamicin and nalidixic acid proved to be the most active against the cultures: 11.9 and 10 per cent of the resistant strains, respectively. The strains of enterobacteria isolated from different sources had a sensitivity to the antibiotics. Multiple antibiotic resistance to at least 5 drugs, variability of the spectra and high resistance were more characteristic of the isolates from the animals. The necessity of a rational use of antibacterial drugs in veterinary is indicated.     

How do we use language? Shared patterns in the frequency of word use across 17 world languages

We present data from 17 languages on the frequency with which a common set of words is used in everyday language. The languages are drawn from six language families representing 65 per cent of the world's 7000 languages. Our data were collected from linguistic corpora that record frequencies of use for the 200 meanings in the widely used Swadesh fundamental vocabulary. Our interest is to assess evidence for shared patterns of language use around the world, and for the relationship of language use to rates of lexical replacement, defined as the replacement of a word by a new unrelated or non-cognate word. Frequencies of use for words in the Swadesh list range from just a few per million words of speech to 191 000 or more. The average inter-correlation among languages in the frequency of use across the 200 words is 0.73 (p < 0.0001). The first principal component of these data accounts for 70 per cent of the variance in frequency of use. Elsewhere, we have shown that frequently used words in the Indo-European languages tend to be more conserved, and that this relationship holds separately for different parts of speech. A regression model combining the principal factor loadings derived from the worldwide sample along with their part of speech predicts 46 per cent of the variance in the rates of lexical replacement in the Indo-European languages. This suggests that Indo-European lexical replacement rates might be broadly representative of worldwide rates of change. Evidence for this speculation comes from using the same factor loadings and part-of-speech categories to predict a word's position in a list of 110 words ranked from slowest to most rapidly evolving among 14 of the world's language families. This regression model accounts for 30 per cent of the variance. Our results point to a remarkable regularity in the way that human speakers use language, and hint that the words for a shared set of meanings have been slowly evolving and others more rapidly evolving throughout human history.     

PHYSICAL STUDIES OF ISOLATED EUCARYOTIC NUCLEI

The degree of chromatin condensation in isolated rat liver nuclei and chicken erythrocyte nuclei was studied by phase-contrast microscopy as a function of solvent pH, K⁺ and Mg⁺⁺ concentrations Data were represented as "phase" maps, and standard solvent conditions selected that reproducibly yield granular, slightly granular, and homogeneous nuclei Nuclei in these various states were examined by ultraviolet absorption and circular dichroism (CD) spectroscopy, low-angle X-ray diffraction, electron microscopy, and binding capacity for ethidium bromide Homogeneous nuclei exhibited absorption and CD spectra resembling those of isolated nucleohistone. Suspensions of granular nuclei showed marked turbidity and absorption flattening, and a characteristic blue-shift of a crossover wavelength in the CD spectra. In all solvent conditions studied, except pH < 2 3, low-angle X-ray reflections characteristic of the native, presumably superhelical, nucleohistone were observed from pellets of intact nuclei. Threads (100–200 A diameter) were present in the condensed and dispersed phases of nuclei fixed under the standard solvent conditions, and examined in the electron microscope after thin sectioning and staining Nuclei at neutral pH, with different degrees of chromatin condensation, exhibited similar binding capacities for ethidium bromide. These data suggest a model that views chromatin condensation as a close packing of superhelical nucleohistone threads but still permits condensed chromatin to respond rapidly to alterations in solvent environment.     

Extracellular release of free fatty acids by rat T lymphocytes is stimulus-dependent and is affected by dietary lipid manipulation

[(3)H]-Arachidonic acid-labelled rat T lymphocytes released radioactivity extracellularly when stimulated by the calcium ionophore A23187 or by monoclonal antibodies to some cell surface structures (CD2, CD5, CD11a, CD18, CD54, T-cell receptor) but not to others (CD49d, CD62L); release was greater with the calcium ionophore. Almost all of the radioactivity released from anti-CD2-stimulated lymphocytes was recovered in the free fatty acid fraction, whereas only about 50 per cent of that released after A23187 stimulation was recovered in this fraction. A23187 stimulation resulted in release of arachidonic acid from a variety of phospholipids (phosphatidylinositol, phosphatidylcholine and perhaps phosphatidylethanolamine), while the monoclonal antibody stimulation released arachidonic acid from phosphatidylinositol and perhaps phosphatidylcholine. Unstimulated lymphocytes released a range of fatty acids extracellularly, with palmitic acid accounting for 35-40 per cent and arachidonic acid for 5 per cent of released fatty acid. Stimulation of lymphocytes with either anti-CD2 or A23187 increased total fatty acid release 1.5- to 1.8-fold. In both cases palmitic acid remained the most predominant fatty acid released but the contribution of arachidonic acid increased. The type of lipid fed to the rats significantly influenced the amount and type of fatty acid released. Fish oil feeding significantly reduced extracellular fatty acid release by stimulated lymphocytes.     

Structural transitions of chromatin induced by netropsin

Structural changes of chromatin induced by interaction of netropsin (Nt) with DNA has been examined by analysis of CD and electromicroscopic measurements. The results demonstrate the existence of a transition from the condensed globular state of chromatin into nucleosomal fibres generated by extremely low Nt concentration up to 1 mole Nt per 200 nucleotides. A second transition occurs at high Nt ratio per DNA phosphate (v' = 0.3). involving disorganization of nucleosomal particles. The interference of the Nt binding with chromatin proteins maintaining the sub- and superstructure will be discussed.     

Use of mildronate in therapy of seroresistant syphilis (serologic and immunologic comparisons

Clinico-serological and immunological examinations were applied to 84 patients suffering from seroresistant syphilis with positive serological reactions within 1.5 to 10 years after termination of the treatment by various methods. The initial diagnosis of recently inflicted secondary syphilis was recorded in 8.9 per cent of the patients. 43.3 and 47.8 per cent of the patients had secondary relapsing syphilis and early occult syphilis, respectively. The percentage of lymphocytes CD3+, CD4+, CD8+ and HLA DR+ was determined in peripheric blood with the monoclonal antibodies OKT-3+, OKT-4+, OKT-8+ and FITC-labeled antimouse Ig. The quantity of IgA, IgG and IgM was determined by radial immunodiffusion. The immunoregulatory index (IRI, the ratio of T-helper/inductor cells and T-suppressor cells/cytotoxics) was calculated. The use of mildronate, a drug developed at the Institute of Organic Chemistry of the Academy of Sciences of the Latvian SSR (Riga), led to normalization of the impaired immunological indices and complete negativation of the serological reactions in the group of the patients with disorders in the immunity status (CD8+ - 33 per cent and IRI - 1.3), p less than 0.01.