Preferential response patterns of cytotoxic T lymphocytes specific for fluorescein isothiocyanate-[FITC] modified autologous cells

Murine cytotoxic responses to TNP-modified syngeneic cells (TNP-self) have been shown to exhibit preferential recognition of K or D end self products encoded by the H-2 complex. In the present study, a number of B10 congenic and recombinant mouse strains were investigated to determine the H-2K and H-2D-restricted FTC-self CTL response patterns, and these were compared with the CTL response patterns obtained for TNP-self. The results indicate that for strains possessing the H-2k,d,h2,h4 haplotypes, respectively, preferential CTL responses were observed against FTC recognized in association with Kk over Dk, Dd over Kd, and Kk over Db. These patterns of preferential CTL responses were the same as those reported for TNP-self as well as several anti-viral CTL responses. In contrast to the results obtained in the B10.A strain, in which Kk preference was observed over Dd for TNP-self CTL, no preferential CTL response was observed when FTC was recognized in association with Kk and with Dd. In this context, it was observed that the CTL response to FTC recognized in association with Dd was particularly strong. This strong D end-associated response was shown to involve D locus products, and no evidence was obtained indicating that L locus self products were involved. These studies are discussed with respect to the possibility that different haptens can be recognized by CTL in association with different self determinants encoded by the same H-2 gene products.     

Incidence of squatting facets on the talus of Indians (Agra region).

230 adult Indian tali (from Agra region) were studied for the incidence of squatting facets. Extension of medial articular facet was observed in all the cases. Medial extension of trochlea was observed in 37% of cases; lateral extension of trochlea in 71.6% of cases, lateral squatting facets in 43.5% of cases and medial squatting in 8.6% of cases. The present data are compared with that of other workers.     

Interference of Mycotoxins with Prenatal Development of the Mouse

The prenatal effects of mycotoxins were investigated in GBA mice given by stomach tube a single dose of either aflatoxin B1 (4 mg/kg), ochratoxin A (8 mg/kg) or zearalenone (20 mg/kg) on pregnancy day 8 or 9. Aflatoxin caused foetal anomalies (exencephaly, open eyes, and protrusion of intestines) after exposure on gestation day 8 but not on day 9. The effects (increased prenatal mortality, reduced foetal growth, and a wide variety of malformations) caused by ochratoxin were much more severe and occurred after treatment on either of the 2 days of gestation. Among the spectrum of malformations, predominantly involving the craniofacial complex and the axial skeleton, the most striking was the total aplasia/dysplasia of the upper facial structures. These defects were always accompanied by exencephaly and anophthalmia. Zearalenone caused no effects. It is concluded that of the 3 mycotoxins screened with the technique used, ochratoxin is the most potent teratogen in mice.     

Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.     

Hexokinase receptors: Preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites

Hexokinase plays an important role in normal glucose-utilizing tissues like brain and kidney, and an even more important role in highly malignant cancer cells where it is markedly overexpressed. In both cell types, normal and transformed, a significant portion of the total hexokinase activity is bound to particulate material that sediments upon differential centrifugation with the crude mitochondrial fraction. In the case of brain, particulate binding may constitute most of the total hexokinase activity of the cell, and in highly malignant tumor cells as much as 80 percent of the total. When a variety of techniques are rigorously applied to better define the particulate location of hexokinase within the crude mitochondrial fraction, a striking difference is observed between the distribution of hexokinase in normal and transformed cells. Significantly, particulate hexokinase found in rat brain, kidney, or liver consistently distributes with nonmitochondrial membrane markers whereas the particulate hexokinase of highly glycolytic hepatoma cells distributes with outer mitochondrial membrane markers. These studies indicate that within normal tissues hexokinase binds preferentially to non-mitochondrial receptor sites but upon transformation of such cells to yield poorly differentiated, highly malignant tumors, the overexpressed enzyme binds preferentially to outer mitochondrial membrane receptors. These studies, taken together with the well-known observation that, once solubilized, the particulate hexokinase from a normal tissue can bind to isolated mitochondria, are consistent with the presence in normal tissues of at least two different types of particulate receptors for hexokinase with different subcellular locations. A model which explains this unique transformation-dependent shift in the intracellular location of hexokinase is proposed.     

Fine needle aspiration cytology of eyelid tumors

Fine needle aspiration (FNA) biopsy was performed on 19 patients with eyelid masses. Six of the patients also had preauricular/submandibular nodal enlargements aspirated. Histopathologic study was performed in ten of the cases. FNA cytology made the diagnosis of an epithelial malignancy in 17 cases (10 sebaceous carcinomas, 4 poorly differentiated carcinomas, 2 squamous cell carcinomas and 1 malignant melanoma). The diagnostic accuracy of FNA cytology in evaluating eyelid masses was thus 89.4%; there were two false-negative cases. All nodal FNA smears revealed metastases of the respective primary tumors. This study indicated that FNA cytology is a simple and efficient method for making the diagnosis of malignancy in eyelid masses, especially in those patients who are not suitable candidates for surgery. Subsequent nodal metastases and tumor recurrence were detected without difficulty using FNA smears.     

Fetal and placental responses to artificially induced hyperthermia in rats.

Pregnant rats were utilized to study the effect of maternal hyperthermia on fetal development. Eight groups of six to eight rats were exposed to ambient temperatures of 43-44 degrees C at various stages of pregnancy. All rats were killed on day 20 of gestation. Edema, microencephaly and microphthalmia followed heat treatment on day 4, 6, or 8 and skeletal defects occurred on day 10 of gestation. Apparently heat stress of dams after day 14 of gestation had little or no effect on embryos. Most placentas from day 6-10 treatment groups were significantly heavier than control and exhibited extensive thickening and necrosis of decidua basalis. Our results suggest that the rat is a useful model for investigating maternal hyperthermia as a possible cause of human placentophathies and fetal retardation.     

Comparison of forest above‐ground biomass from dynamic global vegetation models with spatially explicit remotely sensed observation‐based estimates

Gaps in our current understanding and quantification of biomass carbon stocks, particularly in tropics, lead to large uncertainty in future projections of the terrestrial carbon balance. We use the recently published GlobBiomass data set of forest above‐ground biomass (AGB) density for the year 2010, obtained from multiple remote sensing and in situ observations at 100 m spatial resolution to evaluate AGB estimated by nine dynamic global vegetation models (DGVMs). The global total forest AGB of the nine DGVMs is 365 ± 66 Pg C, the spread corresponding to the standard deviation between models, compared to 275 Pg C with an uncertainty of ~13.5% from GlobBiomass. Model‐data discrepancy in total forest AGB can be attributed to their discrepancies in the AGB density and/or forest area. While DGVMs represent the global spatial gradients of AGB density reasonably well, they only have modest ability to reproduce the regional spatial gradients of AGB density at scales below 1000 km. The 95th percentile of AGB density (AGB₉₅) in tropics can be considered as the potential maximum of AGB density which can be reached for a given annual precipitation. GlobBiomass data show local deficits of AGB density compared to the AGB₉₅, particularly in transitional and/or wet regions in tropics. We hypothesize that local human disturbances cause more AGB density deficits from GlobBiomass than from DGVMs, which rarely represent human disturbances. We then analyse empirical relationships between AGB density deficits and forest cover changes, population density, burned areas and livestock density. Regression analysis indicated that more than 40% of the spatial variance of AGB density deficits in South America and Africa can be explained; in Southeast Asia, these factors explain only ~25%. This result suggests TRENDY v6 DGVMs tend to underestimate biomass loss from diverse and widespread anthropogenic disturbances, and as a result overestimate turnover time in AGB.