Inhibition of angiogenesis and metastasis in two murine models by the matrix metalloproteinase inhibitor, BMS-275291.

BMS-275291 is an p.o. bioavailable, sulfhydryl-based matrix metalloproteinase (MMP) inhibitor currently in clinical development for the treatment of cancer. This inhibitor was designed to potently inhibit MMP activities while minimally affecting those of other metalloproteases (e.g., sheddases) involved in the release of cell-associated molecules such as tumor necrosis factor-alpha, tumor necrosis factor-alpha receptor, interleukin-6 receptor, or L-selectin. In vitro, BMS-275291 is a potent inhibitor (nM) of the activities of MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14. BMS-275291 inhibits tumor growth in a B16BL6 model of experimental metastasis, and in this model, BMS-275291 treatment results in a dose-dependent reduction in the number of lung metastases compared with vehicle controls. BMS-275291 also inhibits angiogenesis in a murine angiogenesis model, where once daily treatment with BMS-275291 results in a dose-dependent inhibition of endothelial cell migration into s.c. implanted Matrigel plugs. Pharmacokinetic studies demonstrated that the plasma concentrations of parent BMS-275291 in mice exceeds the in vitro IC(50) values for MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14 for at least 4 h after the administration of a therapeutic dose of BMS-275291. Taken together, these data demonstrate that BMS-275291 inhibits MMP activities that contribute to tumor metastasis and angiogenesis.

PA26, a novel target of the p53 tumor suppressor and member of the GADD family of DNA damage and growth arrest inducible genes.

Exposure of mammalian cells to hypoxia, radiation and certain chemotherapeutic agents promotes cell cycle arrest and/or apoptosis. Activation of p53 responsive genes is believed to play an important role in mediating such responses. In this study we identified a novel gene, PA26, which maps to chromosome 6q21 and encodes at least three transcript isoforms, of which two are differentially induced by genotoxic stress (UV, gamma-irradiation and cytotoxic drugs) in a p53-dependent manner. A functional p53-responsive element was identified in the second intron of the PA26 gene, in consistance with a mechanism of transcriptional induction of the PA26 gene by p53. No clues to its functions were revealed by sequence analysis, although pronounced negative regulation by serum factors argues for a potential role of PA26 in growth regulation. Immunological analysis suggests that PA26 protein(s) is localized to the cell nucleus. Our results suggest that the PA26 gene is a novel p53 target gene with properties common to the GADD family of growth arrest and DNA damage-inducible stress-response genes, and, thus, a potential novel regulator of cellular growth.

Characterization of a molecular clone of HIV type 2 infectious for Macaca nemestrina.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

Impact of medium-chain monoglycerides on intestinal colonisation by Vibrio cholerae or enterotoxigenic Escherichia coli.

Although a number of studies have shown that various free fatty acids (FFAs) and monoacylglycerides (MGs) have bactericidal properties in vitro, the role of these compounds in vivo has not been determined. This study evaluated the antibacterial properties of medium-chain MGs and FFAs for different bacterial enteropathogens with an in-vitro bacterial killing assay and an in-vivo model of intestinal colonisation. Incubation of test bacteria with medium-chain MGs for 4 h led to 100-10,000-fold reductions in numbers of viable cells of Vibrio cholerae, Salmonella typhi, Shigella sonnei and enterotoxigenic Escherichia coli (ETEC). Lauric acid was the only medium-chain FFA to show comparable in-vitro bactericidal activity. The ability of dietary MGs to reduce or eliminate bacterial colonisation of the intestinal tract was evaluated in mice that were predisposed to bacterial colonisation by treatment with streptomycin (STR+). Mice were treated with streptomycin, challenged intragastrically with V. cholerae or ETEC, and given monocaprin (C10:0 MG) either concurrently or as part of the daily diet. Control mice given STR+ without MGs and challenged with V. cholerae or ETEC showed high numbers of challenged bacteria in gastrointestinal contents by 1 h after administration. Concurrent administration of V. cholerae and C10:0 MG (2.5 mg/ml) caused > 1000-fold reduction in numbers of V. cholerae recovered from the gastrointestinal tracts of STR+ mice. Concurrent administration of C10:0 MG with ETEC did not cause a reduction in the number of viable ETEC present in the intestinal tract of STR+ mice. Administration of C10:0 MG in the diet had no effect on the number of viable V. cholerae or ETEC associated with caecal or ileal tissue of STR+ mice when C10:0 MG in the diet was started 1 day before, the same day, or 2 days after bacterial challenge. Collectively, these results suggested that dietary MGs may prevent intestinal colonisation by bacterial enteropathogens if administered at the time of exposure, but have little effect on established intestinal infections.

Changes in canine postural control after injury to anterior vermal cerebellum.

For a preliminary exploration of cerebellar influences on postural control, dogs were trained to stand quietly erect. Posture was perturbed by sinusoidal movement of the supporting table while the dogs were sighted and while they were blindfolded or stimulated by sinusoidal optokinetic stimuli. Postural responses were evaluated by measuring the relative amplitude of pelvis movement over a series of perturbing frequencies; frequency response descriptions were derived. Lesions of the cerebellar vermis in 3 dogs involved the cortex of lobules V and VI, of lobules IV, V and VI, and of lobules IV--VII plus damage to the medial portion of nucleus fastigius. The two smaller lesions degraded the animal's tracking ability under all three conditions temporarily with extensive but incomplete compensation in 14--21 days. The larger lesion effectively and permanently eliminated the animal's ability to track table or visual field motion. Elimination of visual cues by blindfolding or perturbing the posture with optokinetic stimuli significantly exaggerated the control deficiencies produced by the lesions. It is concluded that the visual input to the cerebellar vermis finds expression through postural control channels.

Postural reactions of dogs to sinusoidal motion in the peripheral visual field.

Dogs were trained to stand quietly on a movable table that was surrounded by a movable roof. The position of the dogs was monitored while the table was stationary and the roof was moved to provide an optokinetic stimulus restricted to the lateral visual fields. Sinusoidal oscillation of the visual surround caused these quietly standing dogs to generate consistent changes in body position. At low frequencies (around 0.1 Hz) the body position changes tended to lead the position of the optokinetic stimulus. At higher frequencies of visual surround oscillation, the dogs' position changes lagged the optokinetic stimulus position by larger amounts as the frequency of motion got higher. These data showing a postural reaction due to motion of the optokinetic stimulus demonstrate a strong visual component of the postural control system.

Modification of the postural response of the normal dog by blindfolding.

1. Normal dogs were trained to stand quietly upon a movable platform. The animals' postural adjustments in response to sinusoidal motion of the platform were assessed in both the normal and the blindfolded condition. The induced postural adjustments were described by the Fourier coefficients for the fundamental of the input together with the coefficients which were integral multiples of the fundamental.2. All of the output variables, including motion at the hind limb joints and of the body, showed modifications when the dogs were blindfolded. The body motion increased in amplitude, as measured by the fundamental Fourier coefficient, and further lagged the input motion. Motion at the knee and phalangeal joints also increased in amplitude and the timing of these movements shifted from the normal in characteristic patterns for different frequencies of input motion.3. The modifications of the postural response due to blindfolding were most pronounced at the lower input frequencies: 0.2-1.0 Hz. At 1.4 Hz or above the fundamental coefficients of most of the output measures resembled those of the normal condition. Even at the higher input frequencies, however, the postural response of the blindfolded dog was more distorted than that of the normal dog.4. It is hypothesized that relatively slow movement of the visual surround provides the dog with information which is essential to normal operations of the postural control system.

Reduction in virus-neutralizing activity of a bovine colostrum immunoglobulin concentrate by gastric acid and digestive enzymes.

Bovine milk immunoglobulin concentrates have been proposed for inducing passive immunity against various enteric pathogens. In vitro digestion studies were conducted to evaluate the effect of gastrointestinal secretions on the virus-neutralizing activity of a concentrate prepared from the colostrum of cows that were immunized with rotavirus. The proteolytic activity of human gastric and duodenal fluid specimens was used to design a two-stage in vitro digestion model with commercial enzymes for estimating the individual impact of pepsin, gastric acid, and select pancreatic enzymes on antirotavirus activity in bovine milk immunoglobulin concentrates. The rotavirus-neutralizing titer of concentrate was decreased by incubation with pepsin at pH 2, a pool of pancreatic enzymes at pH 7.5, or sequential digestion with pepsin (pH 2) and pancreatic enzymes (from initial titer of 55,210 to 2,030, 19,500, and 320, respectively). Reduction in rotavirus-neutralizing titer after gastric-phase digestion was primarily due to acidic conditions and not to proteolytic cleavage by pepsin. Although both trypsin and carboxypeptidase caused significant proteolysis of concentrate during duodenal-phase digestion, only trypsin caused a significant reduction in rotavirus-neutralizing titer. The extent of digestion was the same for concentrate suspended in water or skim milk. The results demonstrate that the biological activity of bovine milk antibodies is reduced by exposure to acid and trypsin in vitro and suggest that neutralization of both gastric acid and pancreatic trypsin may enhance the effectiveness and economic feasibility of passive oral immunoprophylaxis with bovine milk immunoglobulins.

Response of bifidobacterium species to growth promoters in human and cow milk.

We used an in vitro assay to study and compare the growth-promotional activity of protein and nonprotein components in human milk (HM) and cow milk (CM) samples for infant strains of Bifidobacterium species. HM samples varied considerably in growth-promotion activity for Bifidobacterium bifidum var pennsylvanicus, Bifidobacterium infantis, and Bifidobacterium breve. Pooled CM samples showed similar but less variable levels of activity when compared with HM samples. Separation of milk samples by ultrafiltration into protein nitrogen and nonprotein nitrogen (NPN) fractions revealed that the bifidobacteria growth-promotion activity of HM was associated primarily with the NPN fraction, whereas activity in CM whey was found in both protein nitrogen and NPN fractions. Testing of purified CM whey proteins showed that alpha-lactalbumin and lactoferrin were potent growth promoters, showing greater activity for B. infantis and B. breve than for two strains of B. bifidum. Conversely, N-acetylglucosamine and purified gastric mucin were highly active for B. bifidum strains but inactive for other Bifidobacterium species. Collectively, the data indicate that both protein nitrogen and NPN factors in HM and CM promote the growth of bifidobacteria and suggest that Bifidobacterium species differ in responsiveness to protein and oligosaccharide growth promoters.

A predictive model study of the visual contribution to canine postural control.

Dogs were trained to stand on a movable table and their quiet stance was perturbed by osccillation of the table during normal sighted condition and during blindfolded condition. The data formed the frequency response characteristic (describing function) for postural control with and without visual input. A feedback model was tested to assess the effect of visual input during the perturbation of quiet stance. The results of the tests of the model indicate that the effect of a visual input depends on the context of the multiple sensory factors influencing postural control. The effectiveness of the visual input increases if there is a conflict between the visually derived body position cues and the other cues that indicate the orientation of the body.

Optimal and adaptive control in canine postural regulation.

For analytic purposes, dogs trained to stand quietly on an oscillating platform can be likened to a fixed-length inverted pendulum with a point mass. Describing function analysis permitted derivation of torque and error values as functions of phase and gain relative to platform movement. A phase criterion was determined for minimization of either control torque at a given error amplitude or error at a given control torque amplitude. Describing functions for dogs with and without vision approached optimal phase. Stretch reflex control involving proportional-plus-rate feedback is not sufficient to account for the approach to optimal phase. Blindfolded labyrinthectomized dogs did not exhibit optimal behavior and the phase constraint for stretch reflex control was satisfied at most frequencies. The observed behavior is best accounted for by a model involving both otolith and visual feedforward (pursuit-precognitive) control processes. Reductions in phase lag by blindfolded dogs during the first few cycles of platform motion provide evidence of adaptive control.

The postural response of normal dogs to sinusoidal displacement.

1. Normal dogs were trained to adopt a laterally symmetric stance on a horizontal platform. Sinusoidal oscillation of the platform in the cephalocaudal direction caused the dogs to adopt a strategy of response which would keep them from falling down during the period of imposed motion or perturbation.2. A Fourier analysis of the response variables provided a quantitative measure of the distortion in the induced movement at the various hind leg joints and in the motion of the body. Certain aspects of the distortion could be accounted for by recognized random events such as drift and panting. The remainder of the distortion was task related and therefore provided evidence that the dog's postural control system behaved essentially as a non-linear system.3. The motion of the body was less distorted than the motion at the joints. The frequency response of the body motion resembled that of a second order linear system, but the amplitude of the body motion did not vary in constant proportion with changes in the amplitude of the input. Further, the phase relation between body motion and platform motion was not constant when the amplitude of the input was changed. Therefore, it was concluded that the control of the body position was probably non-linearly related to the input disturbance but that the low pass filter characteristics tended to minimize the appearance of distortion in that system output.4. Control of the position of the hind limb is related to the control of the torque generated at the hind limb joints. To the extent that joint angle and change in joint angle are related to the torque at a joint, the distortion of the motion at the joints clearly demonstrates that control of the hind limb during this postural task was non-linearly related to the sinusoidal input.5. The uniformity of the response parameters, as assessed from the Fourier coefficients, indicated that all of the tested dogs adopted the same or nearly the same strategy for solving the problem of adjusting their postural control in response to the perturbation. Therefore, a reasonable hypothesis for future testing is that the central programme which generates this particular postural response will be structured similarly from one dog to the next.

Luxatio erecta humeri. A case report.

Luxatio erecta humeri is a rare type of glenohumeral dislocation. The pathomechanics of this injury involve either direct axial loading on a fully abducted extremity or leverage of the humeral head across the acromion by a hyperabduction force. The clinical presentation of this type of shoulder dislocation is unique, with the affected extremity held rigidly above the head in abduction. Reduction is accomplished by a form of traction-countertraction under intravenous sedation and analgesia. A variety of neurologic and vascular injuries may be associated with luxatio erecta humeri, involving the brachial plexus and axillary artery, respectively. Concomitant fracture of the acromion, clavicle, coracoid, greater tuberosity, and humeral head may also be seen. A computed tomography scan of the case reviewed here revealed a large humeral head defect oriented perpendicular to the classic Hill-Sachs lesion. Luxatio erecta humeri is associated with significant late morbidity, including recurrent dislocation, instability, and adhesive capsulitis.

Growth promotion of Bifidobacterium species by whey and casein fractions from human and bovine milk.

An in vitro assay was used to study the growth-promotional activity of human milk (HM), cow's milk (CM), and whey and casein fractions of HM and CM for five strains of Bifidobacterium species isolated originally from stools of human infants. Whey- and casein-predominant CM-based infant formulas were studied as well. When compared on an equivalent protein basis, the growth promotion activity of HM was greater than that of CM for Bifidobacterium bifidum serovar pennsylvanicus and Bifidobacterium longum but comparable for B. bifidum, Bifidobacterium infantis, and Bifidobacterium breve. Pasteurization of HM and CM resulted in an increase of growth promotion activity for B. bifidum serovar pennsylvanicus and B. bifidum, a decrease for B. infantis, and no change for B. longum and B. breve. The growth promotion activity of HM whey was slightly higher than that of HM casein for four strains of bifidobacteria. When CM casein was a substrate, virtually no growth occurred for B. bifidum serovar pennsylvanicus, B. bifidum, B. infantis, and B. longum. The growth promotion activity of CM whey, however, was similar to that of HM whey. A similar trend was observed for CM-based infant formula. Whey-dominant formulas promoted better growth of B. bifidum serovar pennsylvanicus, B. bifidum, and B. infantis than casein-dominant formulas. The data suggest a direct relationship between amount of whey-specific factors and the ability to promote growth of clinically relevant strains of Bifidobacterium species by HM, CM, and CM-based infant formulas.

Mapping the determinants of human immunodeficiency virus 2 for infectivity, replication efficiency, and cytopathicity.

Human immunodeficiency virus 2 (HIV-2) ISY and the newly derived HIV-2KR are infectious molecular clones that yield viruses differing markedly in their abilities to infect and/or induce syncytia in various T- and monocytoid-cell lines. Chimeric viruses were constructed from these two viral genomes to localize the genetic determinants of some of these properties. Envelope sequences, particularly those spanning the CD4 binding site, appear to be critical for the ability of HIV-2KR to infect MOLT-4 clone 8 and SupT1 cells and to efficiently infect the H9 cell line. On the other hand, multiple determinants may contribute to cytopathicity (gp41 and nef) in H9 cells and replication efficiency in monocytic (THP-1) cells.

The effect of sectioning auditory centrifugal fibers on the cochlear microphonic and action potential in guinea pigs.

This investigation on 8 guniea pigs determined the immediate effect on the cochlear microphonic (CM) and action potential (AP) of ipsilateral sectoning of the autitory centrifugal fibers. An acoustic signal was used to evoke the CM and AP of one ear of guinea pig and the homolateral olivo-cochlear bundle and lateral lemniscus were then sectioned. Differences between pre- and post-section CM and AP were measured. The results demonstrated an increase in the CM and a decrease in the AP in all animals. Speculation with regard to the overall function of the auditory centrifugal system was offered.

Gene regulation by temperature-sensitive p53 mutants: identification of p53 response genes.

The ability of the p53 protein to act as a sequence-specific transcriptional activator suggests that genes induced by p53 may encode critical mediators of p53 tumor suppression. Using a tetracycline-regulated p53 expression system and cDNA library subtraction procedure, we identified several p53-induced gene transcripts in human Saos-2 osteosarcoma cells that are novel on the basis of their size, regulation, and low abundance. Wild-type p53-dependent induction of these transcripts was observed in cells that are growth arrested by p53, as well as in cells that undergo apoptosis upon expression of an inducible wild-type p53 transgene. These results show that p53 activates the expression of numerous response genes and suggest that multiple effectors may play a role in mediating cellular functions of p53.