Vitamin A augments collagen production by corneal endothelial cells

When isolated confluent corneal endothelial cells were cultured in delipidized serum, a marked reduction in collagen production was observed. Supplementation of such cultures with vitamin A as either retinol or retinoic acid at concentrations of 10^?6–10^?7, $\text{M}$ was capable of significantly increasing collagen production. In addition, when cultured in normal (non-delipidized) serum, both retinol and retinoic acid were capable of further increasing collagen production by corneal endothelial cells. Such augmentation of collagen production was relatively specific as total protein synthesis was not altered to the same extent, nor was it merely a reflection of changes in total cell number, as such cell numbers were similar in all treatment groups.     

Changes in hepatic fatty acid synthesis following glucagon injections in vivo

When retinol was incubated in the presence of ATP, UDP-galactose, magnesium ions and Triton X-100, with a whole homogenate of rat thyroid as an enzyme source the formation of a retinol containing phosphate compound was observed besides retinol galactoside described before. This phosphate compound is soluble in chloroform-methanol (3:2, $\text{v}\text{v}$) and can be separated by chromatography on a column prepared with butanol-washed cellulose powder. The isolated compound was retinol pyrophosphate since it contained retinol and phosphate in a molar ratio of 1:2 shown by double isotopic labelling techniques and was found to be free of galactose.     

Preliminary characterization of a small intestinal binding component for retinol and fatty acids in the rat

A component which can bind retinol and fatty acids was detected in the rat's intestinal cell cytosol following intestinal perfusion $\text{in}$$\text{vivo}$ with ³H-all-trans retinol. Following Sephadex G-100 filtration of the cytosol, the void volume concentrate was treated with 2-mercaptoethanol and SDS. Sephadex G-100 filtration of the concentrate disclosed the presence of a cytosol binder of an approximate molecular weight of 12,000–17,000. The binder contained most of the ³H-retinol eluted off the column. $\text{In}$$\text{vitro}$ incubation experiments disclosed that ³H-retinol could be displaced from its bindinf cytosol fraction by the addition of nonradioactive retinol, retinyl acetate, and the fatty acids octanoic, linoleic, and linolenic. Butyric acid addition did not displace ³H-retinol from its binding fraction. The intestinal cytosol binding fraction may be involved in the trans-cytosol transport of lipid compounds from the lipid cell membrane to the intracellular organelles.     

The cell cycle and retinal histogenesis fidget mutant mice.

The autoradiographic method using [³H]thymidine has shown that the autosomal recessive mutant gene fidget (gene symbol fi) prolonging the presynthetic period of the cell cycle in the retinal anlage in homozygotes retards the transition of retinal cells to the differentiated state. Some retinal cells of normal embryos (+/+) start their transition to the differentiated state on the 11th day of embryogenesis, while in $\text{fi}\text{fi}$ embryos this process starts only on the 12th day. An active transition of retinal cells to the differentiated state especially in the peripheral zone of the mutant retina takes place 2 days later as compared to normal embryos. The number of differentiating cells in the retina of mutants at the stages of development studied is considerably lower as compared to the norm. The analysis of the cell cycle parameters in 15-day embryos has shown that in the mutants the retina is less mature as compared to +/+ embryos. The sequence of transition of various cell types to the differentiated state in the retina of $\text{fi}\text{fi}$ embryos is the same as in the norm. Gene fidget seems to interfere with proliferative rather than critical (quantal) cell cycles in the developing mouse retina.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Ontogeny of the retina and optic nerve of Xenopus laevis: IV. Ultrastructural evidence of early ganglion cell differentiation

Ultrastructural evidence indicates that Xenopus retinal ganglion cell axons differentiate early, between stages 28 and 32. Light microscope studies indicated the presence of argryophilic material in the ventral retina and optic stalk of early embryos. Ultrastructural analysis of this region confirmed the presence of axons in the stalk and interstices of ventral retinal cells. Axons containing aligned microtubules and neurofilaments and elongated mitochondria with a paucity of other cell inclusions are found with increasing frequency in the ventral retina from stages 28 through $\text{33}\text{34}$. Central and dorsal regions of the retinas examined show little or no evidence of axons. A discrete, small bundle of axons is found in the optic stalk of stage 28 embryos and by stage $\text{30}\text{31}$ the number of axons in bundles has increased, suggesting early fasciculation. Between stages 28 and $\text{33}\text{34}$ (± 12 hr) extracellular space surrounding early axons diminishes and processes from neuroretinal cells in contact with axons surround developing axon bundles. The evidence presented suggests that axon initiation occurs in stages much earlier than previously reported. Other investigators have failed to detect ganglion cell differentiation prior to stage 32 possibly because they examined regions of the retina with few axons. Thus, experiments which rotate the retina in the orbit may have to be reevaluated since regenerating axons may use previously established pathways to organize and “home in” on tectal target cells.     

Inhibitor of heme synthesis in polycythemic rabbit serum

Sera from hypertransfused polycythemic rabbits were found to significantly inhibit ⁵⁹Fe incorporation into heme in erythroid cells in normal rabbit bone marrow cultures when compared with that of normal serum controls suggesting a higher concentration of this inhibitor in polycythemic serum. This serum inhibitor delayed the time of peak cumulative heme synthesis $\text{in}$$\text{vitro}$ and the delay in peak cumulative heme synthesis was increase with increasing concentrations of polycythemic serum. It is suggested from these studies that this serum inhibitor may be involved in a negative feedback system in the control of erythropoiesis and may act specifically on differentiated nucleated erythroid cells to delay their entry into the cell cycle, consequently inhibiting heme synthesis.     

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. II. Responsible factors for nonspecific destruction

Cell-free supernatants of thoracic duct lymphocyte cultures which were stimulated in vitro by horse serum on syngeneic fibroblast monolayers are demonstrated to be cytotoxic on syngeneic embryonic fibroblasts by means of a direct cell count using microtest plates. Experimental supernatants showed up to 100% suppression of fibroblast growth at $\text{1}\text{3}$ dilution and up to 96% suppression at $\text{1}\text{4}$ dilution as compared to the control supernatants. Evidence is presented indicating that lymphocytes cultured on mosaic monolayers, which were comprised of syngeneic and xenogeneic fibroblasts, were reacting both to xenogeneic cells and horse serum in the medium at the cellular level. A hapten-to-carrier type relationship is suggested between xenogeneic antigen and horse serum. Absence of horse serum in the test cultures using these lymphocytes resulted in the abrogation of nonspecific toxic activity of lymphocytes while the specific activity, though diminished, remained. This again indicates the difference in the mechanisms underlying the specific and nonspecific target cell destruction by T cells.     

Altered DNA sedimentation by the presence of erythrocytes in alkaline sucrose gradient centrifugation

The presence of a relatively small number of red cells was found to affect DNA sedimentation profile of normal lymphocytes and acute leukemia cells, as observed by the alkaline sucrose gradient centrifugation technique coupled with the fluorometric measurement of DNA. Significant alteration was observed at a nucleated cell/erythrocyte ratio of $\text{20}\text{1}$ to $\text{0.2}\text{1}$, resulting in retardation of the $\text{S}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value and the entire sedimentation profile. This effect seemed to be rather specific to erythrocyte lysate, since corresponding amounts of erythrocyte ghost, IgM, bovine serum albumin, and an increased number of nucleated cells did not influence the profile to an appreciable degree.     

Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in $\text{e}\text{e}$ embryos, (2) $\text{e}\text{e}$ larvae from posthatching onward are darker than normal white larvae, and (3) fully grown $\text{e}\text{e}$ animals are sterile.Experiments reported here show that eyelessness in $\text{e}\text{e}$ embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on $\text{e}\text{e}$ blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either $\text{e}\text{e}$ or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation.Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye.The sterility of $\text{e}\text{e}$ larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to $\text{e}\text{e}$ recipients.The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to $\text{e}\text{e}$ recipients.     

Concanavalin A-mediated agglutinability of Balbc3T3 cells grown in media supplemented with different phosphatidylcholines

$\text{Balb}\text{c3T3}$ cells were grown near confluency in media supplemented with 10% fetal calf serum and than exposed for 24 hours to media containing different phosphatidylcholines bound to delipidated fetal calf serum. Compared to cells grown in regular media, 3T3 cells exposed to media containing dioleoyl-phosphatidylcholine dramatically increased their agglutinability by Concanavalin A. Exposure to several other phosphatidylcholines had no effect.     

Regulatory T cells in pregnancy: V. Allopregnancy-induced T-cell-suppressor factor is H-2 restricted and bears la determinants

Spleen cells of allogeneically pregnant mice at the second week of second to fourth parity contain suppressor T cells capable of suppressing an MLR of maternal responders and releasing a soluble factor. The genetic restrictions of this factor have been studied in a $\text{H}\text{-2}{}^{\mathrm{<mtext>k</mtext>}}\text{H}\text{-2}{}^{\mathrm{<mtext>d</mtext>}}$ system using $\text{k}\text{d}$ or $\text{d}\text{k}$ pregnancies, and responder cells from $\text{k}\text{d}$ recombinants. The factor appeared to be IC subregion restricted. Furthermore, its activity could be removed by passage on insolubilized anti-Ia immune serum. Although factor-releasing suppressor cells are antigen specific the factor itself does not appear so, possibly implicating a mixture of fine specificities.     

Determination of the retinal/protein molar ratios for the purple membranes of Halobacterium halobium and Halobacterium cutirubrum

Previously reported values of the retinal/protein molar ratios for the purple membranes from halobacteria are confusing. For $\text{H. halobium}$ values of approximately 1.00 and 0.45 and for $\text{H. cutirubrum}$ values of 0.45 and 0.43 have been published. A redetermination of these ratios in our hands has yielded identical values of 0.99 ± 0.01 for the membranes of both species. This is in agreement with expectations that the ratio be the same for the two species since the similarity of their membrane structure has been established and that every apoprotein molecule be complexed with a retinal since the uniqueness and equivalence of the membrane protein, bacteriorhodopsin, has been demonstrated.     

Stimulation of leydig cell testoterone secretion in vitro and in vivo in hypophysectomized rats by an agonist of luteinizing hormone releasing hormone

Injection of a luteinizing hormone-releasing hormone (LHRH) agonist into 55-day-old male rats which had been hypophysectomized 3 days earlier resulted in a 10- to 30-fold increase in the levels of testosterone in serum and testicular interstitial fluid (IF) in the 4h following injection. The levels achieved were within or above the normal range for intact untreated rats of this age. In similar animals, injection of LHRH agonist also enhanced the serum testosterone response to injected hCG at $\text{1}\text{1}\text{2}\text{h}$, but not at later times after injection, and by 24h reduced IF levels of testosterone suggested that LHRH agonist had begun to inhibit stimulation by hCG. $\text{In vitro}$, dispersed Leydig cells from untreated hypophysectomized rats showed a 2-fold increase in testosterone responsiveness to LHRH agonist when compared to cells from intact rats, and this change was associated with an 80% increase in the number of Leydig cell LHRH-receptors.     

Cross-species axial signaling, with realignment of retinal axes, in embryonic Xenopus eyes

The locus specificities which enable retinal ganglion cells to assemble a topographic retinotectal map are patterned about a pair of (anteroposterior and dorsoventral) retinal axes set down in the early eye bud. We have transplanted a Xenopus laevis eye bud, at stage $\text{23}\text{24}$ when the retinal field is still responsive to the axial signals from the surrounding tissues, into the enucleated eye socket of a comparable stage Ambystoma maculatum embryo. Three days later, when the Xenopus eye had reached early larval stages and was no longer responsive to extraocular signals, the eye was retransplanted into the socket of the Xenopus final carrier embryo. The pattern of retinotectal connections between the eye and the carrier's optic tectum was examined by electrophysiological analysis of the visuotectal projections. The results indicated that many of the retinae had patterned locus specificities about axes derived from the salamander intermediate host. We infer that axial signaling involves fundamental cellular processes which have been highly conserved during evolution.     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

In vitro development of early postimplantation rat embryos

Rat embryos explanted at $\text{7}\text{1}\text{2}$ or $\text{8}\text{1}\text{2}$post coitum were cultured throughout the major stages of organogenesis in a system of rotating bottles containing heat-inactivated, immediately centrifuged (I.C.) serum. About 80% of the $\text{8}\text{1}\text{2}\text{-}\text{day}$ explants and 50% of the $\text{7}\text{1}\text{2}\text{-}\text{day}$ explants developed a blood circulation in the yolk sac; in these embryos, organogenesis and growth rates were similar to those of embryos in vivo. In cultures continued for 4 or 5 days, many of the embryos developed 30–40 somites. There was little difference in the subsequent development of embryos cultured in maternal serum or male serum during the egg-cylinder stage except for a possible decrease in the frequency of normal axial rotation in embryos from the male serum. Development in rotator bottles was much better than in watchglass cultures.     

Cell growth factor activity: New quantitative method in cell culture assay

This quantitative method for the assay of growth factor activity in vitro was developed empirically. Based on the empirical equations, it seems logical to express the cell growth rate by cell duplication frequency, f, and the growth factor activity by the normalized cell growth rate: $\text{1}\text{(T-T}\text{min}\text{)}$. The latter is recommended as the basis of defining an arbitrary unit of growth factor activity. For the in vitro growth factor activity assay, the quantitative method based on the empirical equations has two important features: (1) it appears to express linearly the growth factor concentration in assay, and (2) it is not affected by the initial cell counts and the length of cultivation.The view that these two features overcome the apparent defects associated with the expression systems that use the cell count ratio and net cell count is discussed. The quantitative method based on the empirical equations has been verified with experiments and compared with expression systems using the cell count ratio and net cell count. The experiments, which verified the quantitative method based on the empirical equations, were carried out with four selected cell lines: aortic endothelial cells, mouse 3T3 fibroblasts, Walker 256 carcinoma, and Chang's liver cells, using serum as the source of growth factor(s) for all cell lines. Vitreous, retinal extract, and Walker 256 carcinoma extract were also used for aortic endothelial cells.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.