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.

The p53 tumor suppressor targets a novel regulator of G protein signaling.

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. Herein we report on the identification of a novel p53 target gene (A28-RGS14) that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways.

An infectious chimeric human immunodeficiency virus type 2 (HIV-2) expressing the HIV-1 principal neutralizing determinant.

The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND.

Induction of the growth inhibitor IGF-binding protein 3 by p53.

Transcriptional activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory processes, including cell cycle progression (p21/WAF1), DNA repair (GADD45) and apoptosis (bax). Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.

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.

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.

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.

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line.

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.

The clinician rating of adult communication (CRAC): a clinician's guide to the assessment of interpersonal communication skill.

This paper reports the results of an initial investigation of the psychometric properties of a new clinical marital communication assessment instrument, the Clinician Rating of Adult Communication (CRAC). The sample consisted of 36 marital communication samples from both maritally satisfied and distressed couples. Reliability results indicated that the CRAC demonstrated high levels of internal consistency, test-retest reliability, and interrater agreement. Support for the validity of the CRAC was found in its correspondence with a marital interaction coding system, its relationship to ratings of marital satisfaction, and its concordance with couples' perceptions of their conflict management behavior. Overall, these findings support the conclusion that the CRAC may provide a useful addition to the measurement armamentarium of the marital clinician and researcher.

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.

Comparison of two host cell range variants of feline immunodeficiency virus.

Two molecular clones of feline immunodeficiency virus were compared. The first clone, 34TF10, was from a Petaluma, Calif., isolate; the second, PPR, was isolated from a cat in the San Diego, Calif., area. The cats from which the isolates were obtained suffered from chronic debilitating illnesses. The two molecular clones differed in their in vitro host cell range. The 34TF10 clone infected the Crandall feline kidney and G355-5 cell lines, but replicated less efficiently on feline peripheral blood leukocytes. In contrast, the PPR clone productively infected the primary feline peripheral blood leukocytes but not Crandall feline kidney or G355-5 cells. The 34TF10 and PPR clones had an overall sequence identity of 91%. The env gene was the least conserved (85% at the amino acid level). Additionally, the potential open reading frame for a Tat-like protein, ORF 2, contained a stop codon in the 34TF10 isolate which was not found in the PPR clone. This truncation did not prevent in vitro or in vivo replication of 34TF10. Two splice acceptor sites were identified in the 34TF10 clone. One was 5' to the beginning of the putative tat open reading frame, and the other was 5' to the putative vif product. Both of these acceptor sites were conserved in the PPR clone. The long terminal repeats of the viruses were 7% divergent between the two clones, with a lack of conservation in putative NF-kappa B, LBP-1, and CCAAT enhancer-promoter sites.

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.

A single-stranded nucleic acid binding sequence common to the heterogeneous nuclear ribonucleoparticle protein A1 and murine recombinant virus gp70.

We have used an antisynthetic peptide antiserum to a murine recombinant virus gp70 to probe normal mouse tissues for immunologically related proteins. In addition to cognate gp70s, this antiserum reacts with the heterogeneous nuclear ribonucleoparticle protein A1 by virtue of a 5-amino acid epitope, PRNQG. Further structural similarity is evident both 5' and 3' of this epitope. Since the function of the heterogeneous nuclear ribonucleoprotein particles in the cell is to aid in the stabilization and processing of newly synthesized RNA, we have investigated whether this retroviral sequence exhibits any nucleic acid-binding properties by the same criteria established for the identification of heterogeneous nuclear ribonucleoprotein particles. Analysis of the peptide in a poly(eA) binding assay shows this retroviral sequence to bind with high affinity to single-stranded nucleic acid. This binding occurs in a salt-sensitive manner characteristic of single-stranded nucleic acid-binding proteins. Flanking peptides not containing this sequence generated from either the A1 or gp70 show no ability to bind single-stranded nucleic acids by this assay.

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.

Nucleotide sequence and genomic organization of feline immunodeficiency virus.

An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.

Reference threshold sound-pressure levels for the TDH-50 and ER-3A earphones.

Reference threshold sound-pressure levels were established for a new insert earphone, the ER-3A tubephone, and for the TDH-50 earphone. In test-retest comparisons, the tubephone produced estimates of auditory threshold as reliable as the thresholds produced by the supraaural earphone. Reference thresholds were developed for the two earphones from data contributed by three laboratories. While the TDH-50 data are in good agreement with the provisional ANSI 6-cc coupler reference levels (ASHA, 1982), the ER-3A data are at variance with the manufacturer's provisional recommendation for 2-cc coupler reference thresholds for frequencies below 1 kHz. The differences are attributed to physiologic noise that masked the lower frequency thresholds.