Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450

Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d₂. These experiments yield a value of $\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{D}}\text{= 11}$, a larger effect than has previously been reported for benzylic hydroxylations.     

Utilization of chromosomal markers in cancer research]

The mouse is an unsuitable species for cytogenetical studies to the extent that it has 40 acrocentric chromosomes and the only criterion which could be used to differentiate them is size. We envisaged using in the case of cell grafts donors or recipients of different sex. This technique has, however, been used to a limited extent. Among the other markers which have been utilized, T6T6 of CBA mice must be mentioned. The discovery in 1966 by Léonard and Dekundt of the presence in AKR mice of fusions of the Robertson type (between chromosomes 6 and 15) has generated new interest in experimental work based on the utilisation of chromosome markers. Being interested in the mechanisms of radio-induced leukemia, the authors described how they have introduced the chromosome marker of AKR mice into the C57B1 strain which is very sensitive to the induction of radio-induced leukemias.     

Monoclonal antibodies to ThB detect close linkage ofLy-6 and a gene regulating ThB expression

We have generated three hybridomas producing rat monoclonal antibodies to a surface antigen, ThB, that is shared by murine B lymphocytes and approximately 50 percent of murine thymocytes. These antibodies, produced by immunizations with MOPC-104E cells, appear to recognize the same antigen that was previously detected by rabbit and goat antisera to MOPC-104E cells (Yutoku et al. 1974, Yutoku et al. 1976).Using these antibodies, we have studied a genetic polymorphism that is associated with the level of ThB expression on B lymphocytes but not with the antigen's expression on thymocytes. We present evidence that this trait is controlled by one gene,Thb, which we find to be very closely linked to the gene or genes controlling the Ly-6, Ly-8, DAG, and Ala 1 antigen(s). While the latter four antigens were described as markers on mature T (or activated T and B) lymphocytes, ThB is restricted to immature thymocytes and all B cells. ThB is not expressed on kidney, although some investigators (McKenzie et al. 1977 a, Halloran et al. 1978) report Ly-6 expression on that tissue. SJL/J, C57BL/10JHz, DBA/2J, and AKR/J are among the mouse strains carrying theThb ^h allele, while BALB/cN, CBA/J, C3H.SW/SnHz, and A/J carry theThb ^l allele. The ThB antigen has not yet been identified as a glycoprotein after cell-surface iodination, NP-40 solubilization, and immunoprecipitation.This work was supported in part by grants from the National Institutes of Health (AI-08917, CA-04681, GM-17367).     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Mutations in mice that influence natural killer (NK) cell activity

A series of mutations in mice was tested for splenic NK-cell activity against YAC-1 target cells. Mutations at six loci that reduce NK-cell activity in the homozygous state were identified, including beige (bg), hairless (hr), motheaten (me), obese (ob), steel (Sl) and, to a lesser extent, dominant spotting (W). Motheaten mice displayed the most profound NK-cell deficiency, with NK-cell activity virtually absent. Two mutations, nude (nu) and lymphoproliferation (Ipr), produced elevated NK-cell-mediated lysis. The double homozygous recessivenu/nu bg/bg nude-beige mouse was viable and NK-cell-deficient, with activity slightly higher than that of +/?bg/bg beige littermate controls. Pigmentation mutants related to beige, including pale ears (ep), pearl (pe), and ruby eyes (ru ^2J ) did not dramatically influence NK-cell levels. Unlike the obese gene, other mutations leading to obesity, diabetes (db) and yellow (Asuy), did not impair NK-cell function. The possible site of gene action of these mutants in the NK-cell pathway is discussed.     

A statistical investigation of the time-sampling methods in studying primate behavior

Two commonly used time-sampling techniques in studying animal behavior, namely, fixed interval time point technique and fixed interval time span technique have been investigated, in which their statistical properties and the estimators for frequency and duration have been discussed. Three simple numerical examples have been used to illustrate the calculation of estimates. Finally, a sketch of a stochastic approach to the problem and the resultant estimators are presented, in which all the possible transitions are considered. Therefore, both total frequency and duration of a certain behavior can be estimated by summing up the estimators during each fixed intervening interval with only two end-points being observed.     

The natural occurrence of insulin receptors in groups on adipocyte plasma membranes as demonstrated with monomeric ferritin-insulin

This study was designed to document whether the reported distribution of insulin receptors in small groups of receptor sites randomly distributed in the glycocalyx of adipocytes and isolated adipocyte plasma membranes was a naturally occurring phenomena or due to artifacts. Possible artifacts include: (1) oligomeric forms of ferritin in the ferritin-insulin preparation, (2) an uneven distribution of the glycocalyx on the plasma membrane, or (3) ligand-induced aggregation of occupied receptor complexes. Biogel A 1.5m chromatography of the ferritin-insulin conjugate revealed the ferritin in the ferritin-insulin complex to consist of 55% monomers, 15% dimers, and 30% oligomers. The monomer peak was purified (> 95%) for use in these studies. Cationic ferritin, a glycocalyx marker, when incubated with paraformaldehyde-fixed plasma membranes, was found to be uniformly distributed on the surface of the plasma membrane indicative of uniformly distributed glycocalyx. The ability to demonstrate and inhibit ligand-induced aggregation on the isolated plasma membrane was established with a multivalent ligand, ferritin-concanavalin A. More than 66% of the ferritin-concanavalin A receptors were found in large clusters of 5 or more and 34% as singletons or clusters of up to 4 when incubated at 24°C with fresh membranes. Only 38% of the ferritin-concanavalin A receptors were in large clusters; 62% were singletons or clusters up to 4 on membranes prefixed with paraformaldehyde before incubation. The distribution of the monomeric ferritin-insulin was similar on both adipocytes and purified adipocyte plasma membranes and was consistent with earlier reports with ferritin-insulin. The quantitative distribution of the monomeric ferritin-insulin as singletons or in groups of 2–6 was comparable between the intact cells and isolated membranes incubated at 24°C. The binding of 500 μUnits monomeric ferritin-insulin per ml to the isolated plasma membranes was studied under incubation conditions similar to those used with ferritin-concanavalin A. Under all three conditions, fresh membranes at 24°C and 0–4°C and prefixed membranes at 24°C, the pattern of distribution of the monomeric ferritin-insulin as singletons or groups of 2–6 was identical, indicating that the ligand was not causing aggregation into clusters as did the concanavalin A. Thus, the occurrence of insulin receptors in small groups appears to be a natural phenomenon in the plasma membrane structure of adipocytes.