An ultra-pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting

We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.     

Estimation of the frequency of malformed sperm by slit scan flow cytometry

We have investigated the utility of Slit Scan Flow Cytometry (SSFCM) for measuring the frequencies of malformed sperm heads in control and mutagen treated B6C3F1/CRL mice. In SSFCM, fluorescence profiles of sperm heads stained with the DNA-specific fluorescent dye acriflavine were recorded for sperm flowing lengthwise through a 2.5-microns-thick laser beam. Malformed sperm were detected as having fluorescence profiles that differed substantially from an average fluorescence profile for sperm from untreated mice. Specifically, a sum of squared difference (SSD) value was calculated for the fluorescence profile of each sperm according to the equation (Formula: see text) where c(i) and t(i) are the ith values for the fluorescence profiles from control and test sperm, respectively. Profiles whose SSD exceeded a threshold value of 20 were considered to be from malformed sperm. We measured fluorescence profiles for 500 sperm per mouse from five control mice, five mice injected intraperitoneally daily for 5 days with a total of 375 mg/kg of body weight methyl methane sulfonate (MMS), and for 30 mice injected intraperitoneally daily for 5 days with total doses of procarbazine ranging from 125 mg/kg to 1,250 mg/kg. Sperm were collected from the caudae epididymides 35 days after the last injection. Frequencies of malformed sperm in these samples were also estimated by visual analysis. All samples were analyzed in double blind fashion. The visual and SSFCM malformed sperm frequencies for the samples from control, MMS-treated, and procarbazine-treated mice were correlated (r = 0.83). A dose effect was seen with both the visual and SSFCM estimates for the sperm from the procarbazine-treated mice.     

Tonofilament protein from newborn rat epidermis. Isolation, localization, and biosynthesis of marker of epidermal differentiation.

A novel extraction procedure, previously used on the cell walls of dermatophytes, has been applied to the epidermis of newborn rat. A leucine-rich fraction was isolated which contained over 60% of the total epidermal radioactivity from [3H]leucine in 15 to 20% of the total protein. This fraction was further purified by chromatography in DEAE-cellulose and Sephadex G-200. The protein with the highest specific activity from [3H]leucine was isolated and gave a single band in sodium dodecyl sulfate polyacrylamide gels of molecular weight = 58,000. Antibody to this protein gave a single precipitin band by immunodiffusion and immunoelectrophoresis in agar with the purified protein. This antibody ultrastructurally immunolocalized specifically over tonofilaments in all layers of the epidermis, but showed no reaction in the dermis. The synthesis of this protein in vitro was inhibited by puromycin but not by actinomycin D, suggesting ribosomal synthesis involving a relatively long lived messenger.     

Acetylated methylmannose polysaccharide of Streptomyces.

A polysaccharide composed of 3-O-methyl-D-mannose and D-mannose in a molar ratio of approximately 10:1 and containing 3 to 4 esterified acetyl residues has been isolated from Streptomyces griseus. This acetylated methylmannose polysaccharide (AMMP) is similar to the methylmannose polysaccharide (MMP) of Mycobacterium smegmatis (Gray, G. R., and Ballou, C. E. (1971) J. Biol. Chem. 246, 6835-6842) in its size and composition, the absence of acidic or basic groups, and the lack of a reducing end. It is different, however, in its content of esterified acetyl residues, and it is slightly different in its structure and in its gel filtration properties. The structure of AMMP has been established by proton magnetic resonance spectroscopy, and by combinations of methylation analysis and Smith degradation utilizing non-radioactively labeled polysaccharide and [3H]methyl-labeled polysaccharide obtained from cells grown in the presence of L-[methyl-3H]methionine. It is concluded that AMMP is a linear, nonreducing, neutral polysaccharide composed of a terminal D-mannose residue linked alpha(1 leads to 4) to a chain of 10 consecutive alpha(1 leads to 4)-linked 3-O-methyl-D-mannose residues. The reducing terminal 3-O-methyl-D-mannose residue exists, at least in part, as its alpha-methyl glycoside. The positions of attachment of the ester residues have not been established.     

Nucleotide sequence of a portion of human chromosome 9 containing a leukocyte interferon gene cluster

The nucleotide sequence of a 9937 base-pair portion of human chromosome 9, which contains two complete leukocyte interferon genes (LeIF-L and J), the complete intergenic region, and part of a third related possible pseudogene (LeIF-M), has been determined. The coding regions of the L and J genes are separated by 4363 nucleotides. The coding regions for the putative L and J interferons are 96% homologous and are each surrounded by about 3500 nucleotides of flanking sequences, which are also highly homologous. The L and J genes and their respective flanking sequences comprise a 4000 nucleotide leukocyte interferon gene repeat unit; the L gene repeat unit contains two major insertions not present in the J gene repeat unit. The J gene repeat unit is flanked by sequence features reminiscent of those found surrounding transposable elements. Both the L and J gene repeat units are embedded within sequences that are highly repeated in the human genome. Structural features identified within this portion of chromosome 9 may have been important for the generation of this interferon gene cluster.     

Paraquat ingestion and pulmonary injury.

Ventricular aneurysm is usually a complication of acute transmural myocardial infarction. The development of cardiac aneurysm represents a process of continued thinning and fibrosis of the necrotic tissue of the ventricular wall. Survival allows the development of a solid fibrous scar which of itself does not affect global ventricular function substantially. Hence, ventricular aneurysms can be present for up to 18 years without production of serious symptoms. The cases were reviewed of 45 patients in whom aneurysmectomy and myocardial revascularization were carried out. Surgical mortality was low (6.6 percent, 30 days); survival one year after operation was 76 percent, but at three years had fallen to 47 percent. Cause of late death was dominantly cardiac. In 19 patients post-operative study was done; although graft patency was observed in 98 percent, substantive improvement in ventricular performance was seen in a minority of patients. The outcome in patients with ventricular aneurysm is primarily related to the status of the residual myocardium and to the status of the vessels which supply it. The mechanism of clinical improvement after aneurysmectomy has not been clarified. However, the long-term results appear to be similar to those in patients with extensive myocardial infarction.     

Trace metal modification of immunocompetence. I. Effect of trace metals in the cultures on in vitro transformation of B lymphocytes.

Lymphocytes obtained from the spleen of Balb/C mice have been subjected to transformation by LPS in the presence of varying concentrations of Pb²⁺, Cd²⁺, or Cr³⁺. Both DNA and protein turnover were followed. It was found that Pb²⁺ and Cr³⁺ are mitogenic over a broad range of concentrations, while Cd²⁺ is slightly mitogenic at very low (10^?6M) concentration and rapidly becomes inhibitory of both [³H]TdR and [³H]ALA uptake. Pb²⁺ appears to stimulate the action of LPS, while Cr³⁺ appears to inhibit. Each of the metals protects the lymphocytes from cell death arising from incubation with LPS. The mechanism of the observed changes is as yet obscure.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.