A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

The acute effects of cigarette smoke exposure on experimental skin flaps

Random vascular patterned caudally based McFarlane-type skin flaps were elevated in groups of Fischer 344 rats. Groups of rats were then acutely exposed on an intermittent basis to smoke generated from well-characterized research filter cigarettes. Previously developed smoke inhalation exposure protocols were employed using a Maddox-ORNL inhalation exposure system. Rats that continued smoke exposure following surgery showed a significantly greater mean percent area of flap necrosis compared with sham-exposed groups or control groups not exposed. The possible pathogenesis of this observation as well as considerations and correlations with chronic human smokers are discussed. Increased risks of flap necrosis by smoking in the perioperative period are suggested by this study.     

Sulfate inhibition of molybdate assimilation by planktonic algae and bacteria: some implications for the aquatic nitrogen cycle

Molybdenum is required for both dinitrogen fixation and nitrate assimilation. In oxic waters the primary form of molybdenum is the molybdate anion. Using radioactive [⁹⁹Mol Na₂MoO₄, we have shown that the transport of molybdate by a natural assemblage of freshwater phytoplankton is light-dependent and follows typical saturation kinetics. The molybdate anion is strikingly similar to sulfate and we present data to show that sulfate is a competitive inhibitor of molybdate assimilation by planktonic algae and bacteria. The ability of freshwater phytoplankton to transport molybdate is inhibited at sulfate concentrations as low as 5% of those in seawater and at sulfate: molybdate ratios as low as 50 to 100 times lower than those found in seawater, Similarly, the growth of both a freshwater bacterium and a saltwater diatom was inhibited at sulfate: molybdate ratios lower than those in seawater.The ratio of sulfate to molybdate is 10 to 100 times greater in seawater than in fresh water. This unfavorable sulfate: molybdate ratio may make molybdate less biologically available in the sea. The sulfate: molybdate ratio may explain, in part, the low rates of nitrogen fixation in N-limited salt waters.     

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.     

Correction of lower eyelid ptosis in the anophthalmic orbit: a long-term follow-up

We describe a 10-year review of 53 patients having had correction of lower eyelid ptosis using fascia lata sling suspension by the operation first described in 1973. The overall conclusion is that this continues to be a reliable procedure with a low complication rate. Four major changes relating to operative technique that create a better result are as follows: (1) the surgical correction must begin with a prosthesis that is ideal for the socket; (2) the fascial strip is narrower at 2 mm; (3) the lateral orbital rim burr hole is placed higher; and (4) the passage of the fascial strip is facilitated by the use of Wright's needle. The optimal sequence of operative procedures in the anophthalmic orbit syndrome is (1) correction of enophthalmos and superior sulcus depression, (2) correction of lower eyelid ptosis, and (3) correction of upper eyelid ptosis.     

Dehydrogenase activity and toxinogenesis of a production strain ofCorynebacterium diphtheriae during submerged cultivation

A simple biochemical procedure was obtained for studying metabolism ofCorynebacterium diphtheriae during submerged cultivation based on the modification of the assay of dehydrogenase activity using 2,3,5-triphenyltetrazolium chloride as redox indicator. Results obtained by the estimation of the dehydrogenase activity using TTC are in a good accordance with oxygen consumption assayed manometrically. By following dehydrogenase activity in submerged cultivations of a production strain ofCorynebacterium diphtheriae PW8-Weissensee we found that a massive toxin production is connected with the decrease of the activity of cells. This fall of activity occurs yet during the exponential phase of growth. Especially a sudden fall of succindehydrogenase activity exactly indicates the beginning of a considerable toxin accumulation in the medium. The presence of inhibitory concentrations of iron ions in the medium not only increases the level of dehydrogenase activity but changes its whole kinetics. A retarded and irregular fall of the activity occurs instead of a sharp one typical for good toxin production.     

Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.     

Development ofgusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzyme-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.