Effects of topical administration of 1% brinzolamide on normal cat eyes.

OBJECTIVE: To evaluate the effect of short-term daily topical administration of 1% brinzolamide on the intraocular pressure (IOP) of healthy domestic cats with normotensive eyes and to assess the potential for negative side effects of drug administration. ANIMALS: Twelve privately owned adult domestic cats without physical or ocular abnormalities. PROCEDURE: Normal variation in IOP was determined on day 1. Cats were then treated on days 2-8 with a topical placebo (artificial tear solution) OU q 12 h. On days 9-15 the cats were treated q 12 h with 1% brinzolamide in one randomly selected eye and the placebo in the contralateral eye. All medications (drug and placebo) were administered twice daily at 7 a.m. and 7 p.m. On days 16-22 the cats received no topical medications. IOP, horizontal pupil size in mm and assessment of conjunctival hyperemia were noted OU on days 1, 8, 15 and 22 at 5 time points (9 a.m., 11 a.m., 1 p.m., 3 p.m. and 5 p.m.). Mixed linear regression models were used to compare the IOP of each eye at all time periods for each cat, controlling for age and weight. RESULTS: Mean IOP was not significantly altered in any eye at any time point during the treatment period compared with pretreatment, baseline, or follow-up evaluations. Conjunctival hyperemia and miosis were not detected in either eye at any time point. Conclusions and clinical relevance Short-term q 12 h administration of 1% brinzolamide did not significantly reduce IOP in this small sample population of normotensive cats under these study conditions. No clinically relevant side effects were noted with brinzolamide administration.

Laminitis in a mature elk hind (Cervus elaphus).

Laminitis should be considered as a differential diagnosis in elk presenting with shifting leg lameness, reluctance to move, recumbency and hoof wall ridging. Eliminating the underlying cause and corrective trimming lead to a good prognosis for recovery.

Linearization of two ligand-one binding site scatchard plot and the "IC50" competitive inhibition plot: application to the simplified graphical determination of equilibrium constants.

A simple procedure for linearizing the curved, two ligand-one binding site Scatchard plot resulting from the presence of a constant concentration of competitive inhibitor is proposed; the same procedure may also be applied to the analysis of data derived from the "IC50" competitive inhibition experimental design. Furthermore, a useful generalization of the Cheng-Prusoff correction is presented.

Serum-induced G0/G1 transition in chemically transformed 3T3 cells. Independence of protein synthesis, stable "memory", and enhancement by cycloheximide pretreatment.

Quiescent, chemically transformed (benzo-alpha-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (less than 4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable 'memory' that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this 'memory' probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

Drastically increased expression of MYC and FOS protooncogenes during in vitro differentiation of chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia cells, representing a clonal population of resting B lymphocytes, were induced to differentiate into immunoglobulin-secreting lymphoblasts and plasmablasts by phorbol 12-myristate 13-acetate. The induction resulted in a rapid increase in the molar ratio of secreted/membrane-bound mu-chain mRNA. Immunoglobulin secretion was preceded by a transition of the cells from the G0 to G1 phase of the cell cycle, as indicated by an increase in RNA and protein synthesis, and an overall increase in cellular RNA. The cells, however, became blocked in G1 and did not enter S phase. The expression of MYC and FOS was rapidly induced by the phorbol 12-myristate 13-acetate treatment. The induction of FOS preceded the shift in secreted/membrane-bound mu-chain mRNA molar ratio, while that of MYC occurred concomitantly with the shift, but prior to induction of total RNA synthesis and immunoglobulin secretion. MYC expression remained at a relatively high level during the whole differentiation process. It is thus concluded that a decline of MYC expression is not a prerequisite for differentiation of the chronic lymphocytic leukemia cells. This suggests that MYC expression may play a different role during differentiation of nonproliferating B cells than in the myelomonocytic cell lines HL-60 and U-937, where MYC expression has been reported to decrease during induced differentiation. The results also show that the expression of the MYC and FOS genes does not result in the transition of these cells into the S phase of the cell cycle.

C-myc expression is dissociated from DNA synthesis and cell division in Xenopus oocyte and early embryonic development.

The combined use of a human c-myc probe and of an antibody raised against the human c-myc gene product demonstrated that the Xenopus cells contained a 2.5-kb c-myc transcript and synthesized a c-myc immunoreactive 65-kd polypeptide. In full-grown oocytes, p65c-myc was predominantly located in the nucleus. In non-dividing Xenopus oocytes c-myc mRNA was present at a steady-state level 10(4) times higher than that of growing somatic A6 cells. This very high level of c-myc transcript was reached early in oogenesis and remained constant thereafter. The rate of p65c-myc synthesis also reached high levels, but only in vitellogenic oocytes, suggesting a post-transcriptional control. Although the cell cycle is resumed at a very fast pace in developing embryos, no further increase in total embryonic content of c-myc RNA could be demonstrated up to the swimming tadpole stage. Furthermore, in embryos the rate of synthesis of p65c-myc decreased to a level markedly lower than that of cell cycle-arrested vitellogenic oocytes. This observation suggests that the function of the c-myc gene in the cell cycle may not be implicated directly in sustaining DNA synthesis or mitosis.

Multiple growth-associated nuclear proteins immunoprecipitated by antisera raised against human c-myc peptide antigens.

Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with, particularly for p65, nucleolar enhancement that left an unstained annulus surrounding the nucleolus.

Growth-dependent synthesis of c-myc-encoded proteins: early stimulation by serum factors in synchronized mouse 3T3 cells.

Synthesis of the c-myc gene product was measured during the entire cell cycle of subconfluent mouse 3T3 cells with an antibody raised against a human c-myc synthetic peptide. The antiserum recognized two mouse c-myc-encoded proteins with apparent molecular weights in sodium dodecyl sulfate-polyacrylamide gels of 62,000 and 60,000. Cell-derived p62 was compared with the mouse c-myc gene product synthesized in vitro. Immunoprecipitation, electrophoretic analyses, and peptide mapping provided evidence that p62 is encoded by the mouse c-myc gene. The rate of synthesis of the c-myc proteins was tightly coupled to the cellular growth state of nontransformed A31 3T3 cells, but not to that of their benzo(a)pyrene-transformed derivative (BPA31). Furthermore, the synthesis of the c-myc proteins was stimulated by the exposure of confluent, density-arrested A31 cells to platelet-derived growth factor or fibroblast growth factor. Tightly synchronized cell populations were obtained on the addition of serum factors to subconfluent, serum-deprived A31 cells, and c-myc expression could be monitored for more than one complete cell cycle. One hour after stimulation the steady-state level of the 2.2 kilobase c-myc transcript increased 30-fold relative to that of quiescent cells and decreased thereafter to the level observed during exponential growth. The rate of synthesis of c-myc-encoded proteins was determined by immunoprecipitation after a 2-h labeling period. After an initial sevenfold increase detectable 2 h after serum addition, the rate of synthesis remained constant throughout the rest of the cell cycle. No further changes associated with the late prereplicative period, S phase, G2, or mitosis could be demonstrated. Pulse-chase and long-term labeling experiments revealed different half-lives for the two c-myc-encoded proteins. The half-lives of the c-myc proteins, however, were independent of the cellular growth state. The sustained expression observed throughout the cell cycle suggests that the growth-related function of c-myc may be required during the G0-G1 transition and in all phases of the cycle of the growing cell.

Hydrodynamic and biochemical correlates of the activation of the glucocorticoid-receptor complex.

Possible changes in the size and shape of the glucocorticoid-receptor complex (GRC) following activation remain poorly documented, due to the lability and possible activation of the receptor during the determination of these hydrodynamic parameters. In the present study molybdate was used to stabilize the GRC, thus preventing these uncontrolled transformations. Cytosol prepared from mouse whole brains was incubated for 18 h at 0-2 degrees C with [3H]triamcinolone acetonide (+/- molybdate). Activation was then initiated by incubation at 22 degrees C for variable times and quenched at 0 degree C by adding molybdate. The Stokes radius and sedimentation coefficient of the GRC declined from 77 A and 9.2 S before activation to 58 A and 3.8 S after activation. These measurements remained consistent after recycling GRC between sedimentation and gel filtration procedures and correspond to a 3-fold reduction in the relative molecular mass. The loss and formation of the 297 and 92 kDa species, respectively, after different durations of activation correlated nearly perfectly with increased binding of GRC to DNA-cellulose (DNA-C). The observed size change also correlated well with decreased adsorption to DEAE-cellulose filters (DE-81) and increased adsorption to glass fiber filters (GF/C). The increased adsorption to GF/C may reflect an increase in hydrophobicity which, with extended durations of activation, leads to increased aggregation and reduced binding to DNA-C, but not to a change in adsorption to DE-81. We propose that during activation the 297 kDa form of the GRC splits to form a 92 kDa species that displays an increased affinity for DNA.

Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation.

Cellular oncogenes are DNA sequences implicated in the genesis of cancer, but their functions in the transformation process are not understood. Our experiments provide data linking expression of two well-studied proto-oncogenes, c-myc and c-rasKi, to current knowledge of proliferation control and its perturbation by differentiation and chemical transformation. Growth stimulation of quiescent cells by serum elevates expression of the myc proto-oncogene in Balb/c 3T3 (A31) cells. In two chemically transformed A31 derivatives (BPA31 and DA31), c-myc expression is constitutive. The levels of c-myc mRNA in quiescent and growing transformed cells are nearly the same, and are only slightly elevated compared to the level found in growing A31 cells. By contrast, c-rasKi expression is cell-cycle-dependent in BPA31 cells. The relative abundance of c-rasKi mRNA begins to increase in mid- to late G0/G1. During terminal differentiation of teratocarcinoma stem cells (F9) into nonproliferating endoderm, relative mRNA abundance is diminished more markedly for c-myc than for c-rasKi. These results demonstrate that expression of the myc and rasKi proto-oncogenes is dependent upon the cellular growth state, and that growth control exhibits growth-factor-dependent, cell-cycle-timed oncogene expression. In the case of the BPA31 cells, c-myc is not rearranged, amplified, or overexpressed. However, the oncogene has lost its cycle-dependent regulation in the chemically transformed cells.

Kinetic analyses of activation-induced changes in the hydrodynamic and surface properties of the glucocorticoid-receptor complex in mouse brain.

Unactivated, molybdate-stabilized, [3H]triamcinolone acetonide-labeled, glucocorticoid receptors from mouse whole brain were activated by removal of the molybdate and incubation at 22 degrees C for 1.5 to 24 min and then rapidly quenched at 0 degrees C with molybdate. The loss of the 9.2 S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8 S (activated) form displayed first-order kinetics with a half-time of less than two min. The increase in the 3.8 S form correlated nearly perfectly with an increased binding to DNA-cellulose, and with a decreased and increased adsorption to DEAE-cellulose and glass fiber filters, respectively. The changes in adsorption to these filters, which occurred at a faster rate than did the changes in binding to DNA-C, are thought to reflect an increase in the relative number of positive charges and hydrophobic groups on the surface of the activated complex.

Cellular oncogenes, growth factors, and cellular growth control.

In this article we relate the functioning of oncogenes, particularly myc and ras, to current ideas regarding regulation of mammalian cell growth by growth factors. Assuming the genetic basis of transformation to be alterations of several proto-oncogenes, the mechanisms by which transformation could diminish growth control are numerous. The oncogene could interact with a growth factor in several ways. Mutations could alter the quantity of an oncogene's product or its quality through primary structure or covalent modifications such as phosphorylations. Oncogenes could code for a receptor for a growth factor. Various alterations parallel to the above set could then affect growth factor function via receptor changes (including abolished requirement for the factor). Some oncogenes might operate during the chain of intracellular events that must follow growth stimulation. Introduction of such an oncogene (e.g., the coding region for a DNA virus T antigen) would bypass requirements for both growth factors and receptors. Various observations regarding the oncogenes, cell cycle-timed events, and growth factors have been presented in a way we hope will suggest experiments designed to provide a basis for understanding growth regulation at the genetic, biochemical, and cellular levels in normal cells and tumorigenic cells, which have deranged growth regulation.

Effect of lesions of the locus coeruleus complex on the circadian rhythm of plasma corticosterone in the mouse.

An apparently transient elevation of basal morning (08.00 h) plasma corticosterone levels in male mice was found 48 h after bilateral electrolytic lesions of the brainstem locus coeruleus complex but was not observed 6 weeks after lesioning.

Sectioning of polyacrylamide slab gels with a modified small animal stereotaxic instrument.

A simple and inexpensive modification of the Kopf model 900 small animal stereotaxic instrument allows it to be used temporarily as a precision polyacrylamide slab gel slicer.

Inhibition of testosterone 5alpha-reductase activity and growth of accessory sex tissues in castrated male mice.

The steroid 4-androsten-3-one-17beta-carboxylic acid (17betaC) reduced the growth-promoting actions of testosterone, but not those of DHT in accessory sex tissues of castrated mice. The 5alpha-reduction of testosterone to DHT in these tissues was also reduced by 17betaC treatment, suggesting that DHT formation is a required step in the mechanism of action of testosterone.

Estrogen-induced sexual receptivity and localization of 3H-estradiol in brains of female mice: effects of 5 alpha-reduced androgens, progestins and cyproterone acetate.

Sexual receptivity induced in ovariectomized CD-1 mice with chronic daily administration of estradiol benzoate (E2 B) was blocked by concurrent administration of the 5 alpha-reduced androgen, dihydrotestosterone (DHT). Receptivity was restored in these females with progesterone-, but not with dihydroprogesterone-priming 6 hr prior to testing. Delaying the DHT injections until 12 hr after the E2 B injections greatly reduced its inhibitory properties. Receptivity in E2 B-primed females was also blocked by concurrent treatment with cyproterone acetate and 3 alpha-, but not 3 beta-adrostanediol. Pretreatment with DHT, or 3 alpha- or 3 beta-androstanediol failed to consistently affects 3H-estradiol accumulation in crude nuclear and supernatant fractions from brain and pituitary.

PIP: Sexual receptivity induced in ovariectomized CD-1 mice with chronic daily administration of estradiol benzoate (EB; 2 mcg) was investigated and localization of tritiated estradiol in brains of these mice attempted. After 10 days of EB priming dihydrotestosterone (DHT) was given (1 mg/day) and again tested for receptivity. This was followed by further testing after DHT had been stopped. In a 2nd experiment the effect of varying dose and time of administration of DHT on estrogen-induced receptivity was attempted. Dosage of DHT was varied from 500 mcg to 1 mg and time of injection was varied from concurrent with EB injection to 12 hours after. In a 3rd study the effects of DHT, cyproterone acetate (CA), and 3alpha- or 3beta-androstanediol were examined with relation to receptivity. A dosage schedule designed to inhibit receptivity was used. Finally, the effects of DHT and 3alpha- and 3beta-androstanediol on tritiated estradiol localization in brain and pituitary was investigated. Estrogen-induced receptivity was blocked by concurrent administration of the 5aplha-reduced androgen, and DHT. Receptivity was restored in these females with progesterone. However, priming with dihydroprogesterone 6 hours prior to testing was ineffective in restoring receptivity. Delayed DHT injections greatly reduced inhibitory properties. Receptivity was also blocked by concurrent treatment with CA and 3alpha-androstanedio. 3beta-androstanediol was ineffective in blocking receptivity. Pretreatment with DHT or 3alpha- or 3beta-androstanediol failed to affect tritiated estradiol accumulation consistently in crude nuclear and supernatant factions from brain and pituitary.