Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts

Summary We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11–13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an¹²⁵I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4, using a high or a low protein dose (500 µg or 5 µg). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as thecoefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-µg protein doses had substantially lower CV values, 0.327±0.027, than did tumors from animals given 5-µg protein doses, 0.458±0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.     

Factors associated with intestinal amyloidosis in pigtailed macaques (Macaca nemestrina).

Factors associated with intestinal amyloidosis in pigtailed macaques (Macaca nemestrina) were studied in 74 cases at the Washington Regional Primate Research Center. The medical records of monkeys during the 5-year period from 1983 to 1988 were analyzed to determine the age at death, age at first episode of diarrhea, number of episodes of diarrhea, episode and cumulative duration of diarrhea, and etiologies of diarrhea. Univariate analysis, using one control for each case, indicated that only episode duration was related to intestinal amyloidosis. Affected monkeys had significantly longer mean episode durations of diarrhea. None of the etiologies examined--bacteria, protozoa, fungi, and simian retrovirus--were significant risk factors for amyloid deposition in the intestinal tract.     

The B95-8 isolate of Epstein-Barr virus arose from an isolate with a standard genome.

Blot hybridization studies revealed that the deletion which characterizes the DNA from the B95-8 strain of Epstein-Barr virus was not present in the virus from which the B95-8 strain was derived (883L). The deletion event must have occurred during establishment of the B95-8 cell line or very soon afterward, since the deletion was present in Epstein-Barr virus DNA from a cell line established with B95-8 virus soon after it became available. The presence of the deletion correlates with decreased expression of the gp220 viral envelope glycoprotein.     

Nuclear shuttling of yeast scaffold Ste5 is required for its recruitment to the plasma membrane and activation of the mating MAPK cascade.

Localization of Ste5 to GP at the plasma membrane is essential for transmission of the pheromone signal to associated MAP kinase cascade enzymes. Here, we show that this crucial localization requires prior shuttling of Ste5 through the nucleus. Ste5 shuttles through the nucleus constitutively during vegetative growth. Pheromone enhances nuclear export of Ste5, and this pool translocates vectorially to the cell periphery. Remarkably, Ste5 that cannot transit the nucleus is unable to localize at the periphery and activate the pathway, while Ste5 with enhanced transit through the nucleus has enhanced ability to localize to the periphery and activate the pathway. This novel regulatory scheme may ensure that cytoplasmic Ste5 does not activate downstream kinases in the absence of pheromone and could be applicable to other membrane-recruited signaling proteins.     

Establishing the principles of recognition in the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa

The protein-based molecular recognition of the adenine ring has implications throughout biological systems. In this paper, we discuss the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa], which serves as an excellent model system for proteins that bind the adenine ring. This enzyme employs a hydrogen-bonding network involving water molecules along with enzyme backbone/side-chain atoms and a pi-pi stacking interaction to recognize the adenine ring. Our approach utilized site-directed mutagenesis, adenosine analogues and a variety of biophysical methods to probe the contacts in the adenine-binding region of APH(3')-IIIa. The results point to the polar nature of an adenine-Met90 contact in this binding pocket and the important role that Met90, the "gatekeeper" residue in structurally similar Ser/Thr protein kinases, plays in adenine binding. The results also suggest that small changes in the structure of the adenine ring can lead to significant changes in the ability of these analogues to occupy the adenine-binding region of the enzyme. Additional computational experiments indicate that both size and electronic factors are important in the binding of aromatic systems in this interaction-rich pocket. The principles governing adenine recognition established in this study may be applied to other protein-ligand complexes and used to navigate future studies directed at discovering potent and selective inhibitors of APH-type enzymes.     

Widespread recombinase expression using FLPeR (flipper) mice

As conditional genetic strategies advance, the need for multiple site-specific recombinase systems has emerged. To meet this need in part, we have targeted the constitutive ROSA26 locus to create a mouse strain with generalized expression of the enhanced version of the site-specific recombinase FLP (FLPe). This strain is designated FLPeR ("flipper"). Using this strain, extensive target gene recombination can be achieved in most tissue types, including cells of the developing germ line. FLPeR mice therefore serve two important functions: as a source of many different FLPe-expressing primary cell lines and as a deleter strain. Moreover, because the FLPeR mouse is a 129-derived strain, a 129 genetic background can be preserved when crossed to most ES cell-derived mice. This enables conditional genetic alterations to be maintained on a standard background, a feature important for obtaining reproducible results and genetically defined controls.     

Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae haemagglutinating pili

Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well-characterized forms of pili, H. influenzae haemagglutinating pili are two-stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone-subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB-HifA periplasmic complexes, whereas mutations at the -14 glycine in HifA had no effect on HifB-HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD-PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure-function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.     

Parameterization of the shape of intracranial saccular aneurysms using Legendre polynomials

Our recent studies of the nonlinear mechanics of saccular aneurysms suggest that it is unlikely that these lesions enlarge or rupture via material (limit point) or dynamic (resonance) instabilities. Rather, there is a growing body of evidence from both vascular biology and biomechanical analyses that implicate mechanosensitive growth and remodeling processes. There is, therefore, a pressing need to quantify regional multiaxial wall stresses which, because of the membrane-like behavior of many aneurysms, necessitates better information on the applied loads and regional surface curvatures. Herein, we present and illustrate a method whereby regional curvatures can be estimated easily for sub-classes of human aneurysms based on clinically available data from magnetic resonance angiography (MRA). Whereas Legendre polynomials are used to illustrate this approach, different functions may prove useful for different sub-classes of lesions.