Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.     

Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching

Brain tubulin has been conjugated with dichlorotriazinyl- aminofluorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) that DTAF-tubulin polymerizes like control tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (less than 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into living cells) will retain full polymerization activity. Slow bleaching (approximately 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-tubulin, including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-tubulin, however, DTAF- microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. These results suggest that DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.     

Ultrastructural detection of the onset of pituitary thyrotroph sensitivity to lowered thyroid hormone concentrations in the fetal sheep

The goitrogen methylthiouracil was administered orally to pregnant ewes of known gestational ages to induce hypothyroidism in both mother and fetus. Developing pituitary thyrotrophic cells were studied using electron microscopy to detect the earliest gestational age at which morphological changes occurred in response to lowered plasma thyroid hormone concentrations. At 50 days of gestation, the pituitaries of fetuses exposed to the goitrogen were indistinguishable from untreated control glands. However, at 58 days and subsequent ages, "thyroidectomy' cells were observed in pituitaries of all hypothyroid fetuses. These findings indicate that fetal sheep pituitary thyrotrophs are sensitive to lowered thyroid hormone concentrations by 58 days of gestation, suggesting that thyroid-thyrotroph interaction exists at this early stage of development.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Secretory IgA in Urinary Tract Infections

Secretory IgA, measured by radial immunodiffusion, was compared in the urine of children with chronic and recurrent non-obstructive urinary tract infections with that in normal children. IgA, IgG, and IgM were also measured. Absent and low levels of IgA(s) were found in both groups; however, the mean levels of IgA(s) were significantly higher in the infected group compared with normals—3·3 to 0·78 mg./24 hours, respectively. Secretory IgA was found to be locally produced in the bladder. It is suggested that IgA(s) levels reflect an antibody response to infection.     

Interdependence of inoculum size and trace element supply in growth of Aspergillus oryzae

Summary Further to earlier reports indicating that the effects of inoculum size on growth of Aspergillus oryzae in synthetic media are abolished by certain trace element compositions, it has now been shown that those effects can be made more pronounced, but can also be reversed, by varying the balance of available trace elements.Addition of Ca to substrate A ₁ increases the usual effects of inoculum size whereas Cu abolishes them with a tendency for reversal.Supplementation of substrate A ₁ with suitable amounts of ethylenediaminetetraacetic acid results in a stronger inhibition of small-inoculum cultures than large ones, thus increasing the usual effects of inoculum size. These effects are reversed if the sugar of substrate A ₁ is purified by Al(OH)₃ co-precipitation method, but only if the treatment is carried out at neutral or alkaline reaction. This reversal of the phenomena is due to an almost complete absence of Zn after purification and presence of Fe in substrate A ₁.The Al(OH)₃ treatment of the sugar results in substrates so poor in Zn that addition of as little as 5 g ZnSO₄ · 7 H₂O/l has an effect. This influence, however, results in an inhibition of the growth rate, and higher concentrations of Zn are required in order to obtain stimulation with both large and small-inoculum cultures.The above-mentioned reversal of the phenomena was abolished in a substrate with slightly caramelized glucose.

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Zusammenfassung Diese Arbeit ist eine Erweiterung vorhergehender Berichte über den Einfluß der Größe der Impfmenge auf das Wachstum von Aspergillus oryzae, wo u. a. festgestellt wurde, daß die beobachteten Effekte durch bestimmte Spurenelement-Zusammensetzungen aufgehoben werden.Es wurde nun festgestellt, daß Zugabe von Ca zu Substrat A ₁ die gewohnten Effekte erhöht, während Cu diese verringert mit einer Tendenz zur Umkehrung.Werden geeignete Mengen Äthylendiamintetraessigsäure zu Substrat A ₁ zugegeben, so werden Kulturen, die aus kleiner Impfmenge erwachsen sind, stärker in ihrer Wachstumsgeschwindigkeit und maximaler Ausbeute an Mycel gehemmt als Kulturen von großer Impfdichte, was einer Verstärkung der gewohnten Effekte gleich kommt. Diese Effekte werden umgekehrt, wenn die Glucose des Substrates A ₁ durch Al(OH)₃-Fällung gereinigt wird, jedoch nur wenn diese Behandlung bei neutraler oder alkalischer Reaktion erfolgt. Diese Umkehrung ist auf nahezu vollständige Entfernung von Zn und gleichzeitige Anwesenheit von Fe in Substrat A ₁ zurückzuführen.Schon Zugabe sehr kleiner Mengen Zn (5 g ZnSO₄ · 7 H₂O/l) zum gereinigten Substrat übt eine Wirkung aus, welche aber in einer Hemmung der Wachstumsgeschwindigkeit besteht. Höhere Zn-Konzentrationen sind nötig, um das Wachstum der Kulturen von großen und kleinen Impfmengen zu fördern.Die vorerwähnte Umkehrung der Phänomene wurde aufgehoben, wenn ein Substrat mit leichter Caramelisierung der Glucose angewendet wurde.