Dialysable leucocyte extract (DLE) reduces lipopolysaccharide- induced tumour necrosis factor secretion in human leucocytes

Dialysable leucocyte extract (DLE), obtained from lysed leucocytes, provide clinical effectiveness in a broad spectrum of diseases. Tumour necrosis factor (TNF) is raised in AIDS patients leading to an increase in human immunodeficiency virus (HIV) replication in vitro [1,2], whereas progression to AIDS in asymptomatic HIV infected individuals is retarded under treatment with DLE. In the present study we tested the DLE effect in vitro on both TNF biological activity (cytotoxicity) in L929 cells and its induction by lipopolysaccharide (LPS) in human monocytes as well as in whole blood from healthy donors. When monocytic cells were simultaneously exposed to LPS and DLE during a period of 5 1/2 hours, the induction of TNF was strongly diminished. The same inhibitory effect of DLE on TNF induction was observed when LPS was added to the culture medium prior to DLE. No significant effect of DLE on TNF-mediated cytotoxicity, even in the presence of the highest concentrations of DLE tested, was detected. DLE treatment of whole human blood regulates responses to LPS: simultaneous in vitro exposure to endotoxin provokes a remarkable decrease (4- and 1.6-fold) of TNF release. In pre-incubation experiments, TNF production was largely reduced or completed abrogated. These results could, in part, explain the in vivo observed effect, when under treatment with this extract, the progression to AIDS of HIV-infected individuals was retarded. The results suggest that ‘natural’ substances like DLE may be important immunomodulators in inflammatory diseases.     

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

A Technique for Fluorescence Microscopy in Semithin Sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.     

A technique for fluorescence microscopy in semithin sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase

The specific chemical modification by sodium cyanate of highly reactive cysteine residues at pH 7.5 in pig kidney fructose 1,6-bisphosphatase results in the reversible loss of activation of the enzyme by monovalent cations. No loss of activation by potassium ions occurs when modification is carried out in the presence of fructose 2,6-bisphosphate. The effect of Mg2+ on native and cyanate-modified enzyme activities implicates the above cysteine residue as being directly linked to the inhibition by both the divalent cation and fructose 2,6-bisphosphate. Incorporation of [14C]cyanate to the enzyme shows that the blockage of two reactive residues per tetramer is sufficient to eliminate the activation of the enzyme by K+.     

Prepubertal rat ovary: hormonal modulation of beta-adrenergic receptors and of progesterone response to adrenergic stimulation

We have previously shown that, in the rat, ovarian beta-adrenergic receptor content varies during the time of puberty, with values first increasing and then decreasing abruptly on the afternoon of the first proestrus, i.e., at the time of the preovulatory surge of gonadotropins and prolactin (Prl). In the present study, experiments have been conducted to determine: 1) if hormones other than follicle stimulating hormone (FSH) that are known to be involved in regulating prepubertal ovarian function can mimic the facilitatory effect of FSH on progesterone (P) response of granulosa cells to beta-adrenergic stimulation; 2) if beta-adrenergic receptor content of granulosa cells is under hormonal regulation; and 3) whether the facilitatory effect of hormones on the P response to beta-adrenergic stimulation is due to an increased cyclic AMP response to receptor activation. A 48-h in vitro preexposure of granulosa cells from juvenile, 29-day-old ovaries to the pituitary hormones Prl, luteinizing hormone (LH), or FSH showed that only the latter was able to facilitate the subsequent P response to Zinterol, a beta2-adrenergic agonist. Follicle-stimulating hormone also increased basal P release. Of the two nonpituitary hormones examined, the luteinizing hormone-releasing hormone (LHRH) agonist D-(Ala6,Pro9)-LHRH-ethylamide (LHRH-A) failed to affect P responsiveness, whereas corticosterone enhanced both basal P release and P response to Zinterol. This effect was less pronounced than that of FSH. Luteinizing hormone, Prl and corticosterone decreased beta-adrenergic receptor content to different extents, with corticosterone being the most effective and LH the least (50% and 15% decrease, respectively); LHRH-A was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.