Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

The $5 man: the underground economic response to a large cigarette tax increase in New York City.

OBJECTIVES: We examined the mechanisms by which living in a disadvantaged minority community influences smoking and illegal cigarette sale and purchasing behaviors after a large cigarette tax increase. METHODS: Data were collected from 14 focus groups (n=104) that were conducted during the spring of 2003 among Blacks aged 18 years and older living in New York City. RESULTS: A large tax increase led to what focus group participants described as a pervasive illegal cigarette market in a low-income minority community. Perceived pro-smoking community norms, a stressful social and economic environment, and the availability of illegal cigarettes worked together to reinforce smoking and undermine cessation. CONCLUSIONS: Although interest in quitting was high, bootleggers created an environment in which reduced-price cigarettes were easier to access than cessation services. This activity continues to undermine the public health goals of the tax increase.

Extinction of LINE-1 activity coincident with a major mammalian radiation in rodents.

LINE-1 transposable elements (L1s) are ubiquitous in mammals and are thought to have remained active since before the mammalian radiation. Only one L1 extinction event, in South American rodents in the genus Oryzomys, has been convincingly demonstrated. Here we examine the phylogenetic limits and evolutionary tempo of that extinction event by characterizing L1s in related rodents. Fourteen genera from five tribes within the Sigmodontinae subfamily were examined. Only the Sigmodontini, the most basal tribe in this group, demonstrate recent L1 activity. The Oryzomyini, Akodontini, Phyllotini, and Thomasomyini contain only L1s that appear to have inserted long ago; their L1s lack open reading frames, have mutations at conserved amino acid residues, and show numerous private mutations. They also lack restriction site-defined L1 subfamilies specific to any species, genus or tribe examined, and fail to form monophyletic species, genus or tribal L1 clusters. We determine here that this L1 extinction event occurred roughly 8.8 million years ago, near the divergence of Sigmodon from the remaining Sigmodontinae species. These species appear to be ideal model organisms for studying the impact of L1 inactivity on mammalian genomes.

The end of the LINE?: lack of recent L1 activity in a group of South American rodents.

L1s (LINE-1: Long Interspersed Nuclear Element 1) are present in all mammals examined to date. They occur in both placental mammals and marsupials and thus are thought to have been present in the genome prior to the mammalian radiation. This unusual conservation of a transposable element family for over 100 million years has led to speculation that these elements provide an advantage to the genomes they inhabit. We have recently identified a group of South American rodents, including rice rats (Oryzomys), in which L1s appear to be quiescent or extinct. Several observations support this conclusion. First, genomic Southern blot analysis fails to reveal genus-specific bands in Oryzomys. Second, we were unable to find recently inserted elements. Procedures to enrich for young elements did not yield any with an intact open reading frame for reverse transcriptase; all elements isolated had numerous insertions, deletions, and stop codons. Phylogenetic analysis failed to yield species-specific clusters among the L1 elements isolated, and all Oryzomys sequences had numerous private mutations. Finally, in situ hybridization of L1 to Oryzomys chromosomes failed to reveal the characteristic L1 distribution in Oryzomys with either a homologous or heterologous probe. Thus, Oryzomys is a viable candidate for L1 extinction from a mammalian host.

An ancient retrovirus-like element contains hot spots for SINE insertion.

Vertebrate retrotransposons have been used extensively for phylogenetic analyses and studies of molecular evolution. Information can be obtained from specific inserts either by comparing sequence differences that have accumulated over time in orthologous copies of that insert or by determining the presence or absence of that specific element at a particular site. The presence of specific copies has been deemed to be an essentially homoplasy-free phylogenetic character because the probability of multiple independent insertions into any one site has been believed to be nil. Mys elements are a type of LTR-containing retrotransposon present in Sigmodontine rodents. In this study we have shown that one particular insert, mys-9, is an extremely old insert present in multiple species of the genus Peromyscus. We have found that different copies of this insert show a surprising range of sizes, due primarily to a continuing series of SINE (short interspersed element) insertions into this locus. We have identified two hot spots for SINE insertion within mys-9 and at each hot spot have found that two independent SINE insertions have occurred at identical sites. These results have major repercussions for phylogenetic analyses based on SINE insertions, indicating the need for caution when one concludes that the existence of a SINE at a specific locus in multiple individuals is indicative of common ancestry. Although independent insertions at the same locus may be rare, SINE insertions are not homoplasy-free phylogenetic markers.

Validity of urinary catheter specimen for diagnosis of urinary tract infection in the elderly.

Twenty elderly nursing home patients with long-term indwelling bladder catheters were studied to evaluate the validity of the microbiology of urine samples obtained from catheters that had not been changed for at least 30 days. Paired urine samples from "old" catheters and newly inserted catheters were compared for quantitative and qualitative microbiology. Urine microbiology for old catheters was highly sensitive but had poor specificity.

The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori.

The genes encoding phytase (EC 3.1.3.8) and pH 2.5-optimum acid phosphatase (EC 3.1.3.2) have been cloned and sequenced from Aspergillus niger var. awamori. The translated nucleotide sequences yielded polypeptides of 467 and 479 amino acids (aa) for phytase and acid phosphatase, respectively. The genes were isolated using oligodeoxyribonucleotide probes based on the aa sequences of the purified proteins. Recombinant A. niger var. awamori strains carrying additional copies of the gene sequences demonstrated elevated enzyme activities.

Isolation of markers from recently transposed LINE-1 retrotransposons.

Many previous techniques for the isolation of endogenous retroelements such as LINE-1 retrotransposons have produced major sampling bias or required laborious procedures. These problems led to the isolation of only older elements in some cases. In other cases, specialized systems were required for the isolation of recently transposed elements. We report here a system for the easy isolation of markers from a wide range of LINE-1 elements and the screening of recently transposed elements from that population. This is accomplished by the use of PCR with degenerate primers specific for conserved regions of the reverse transcriptase gene, a modified screening vector, and a refined blue/white colony assay that screens for amplified DNA containing open reading frames. This method should be applicable to searches for endogenous retroviruses.

Characterization of monoclonal antihuman small bowel and colon specific mucin antibodies.

Three human small bowel and colon mucosal specific monoclonal antibodies with distinct morphologic and electrophoretic characteristics were generated by fusion of immunized Balb/c spleen cells and murine plasmacytoma cells. Morphologic specificity by indirect immunofluorescence (IIF) revealed three antibody binding patterns corresponding to villus surface (TP-NG-43), goblet cell apical granules (TP-NG-2), and a combined surface/goblet cell apical granule antibody (TP-NG-20). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) produced three distinct electrophoretic migration patterns. These antibodies reacted with very high molecular weight determinants: TP-NG-2, one band greater than 400 kD; TP-NG-20, two bands corresponding to 370-400 kD; and TP-NG-43, two bands in the 350-400-kD range with smaller bands in the 50-94-kD range. Cross-reactivity with various other human organ systems was evaluated by indirect immunofluorescence and SDS-PAGE electrophoresis with Western blotting. By IIF, all three monoclonal antibodies reacted very strongly with components of gastric mucosa. Weak cross-reactivity was seen with colon, rectum and mucin-producing adenocarcinoma of the colon. No cross-reactivity was observed by IIF with other mucin-containing and non-mucin-containing tissues. However, cross-reactivity with gastric mucin was not detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antibody reactivity with mucin was confirmed by purifying various regional gastrointestinal mucins and by subsequent testing by ELISA. Monoclonal antibody affinity columns were prepared and evaluated. The utility of these methods will allow for further definition of important goblet cell mucin glycoprotein characteristics and isolation of mucin subpopulations.

Survival strategies in tropical rain pools.

Adaptations of three larval diptera in central Africa, each inhabiting rain-filled rock pools of characteristic duration, have been studied by field observation and experiment.The duration of the pool is important in determining which of the three species is present. Large pools, lasting a few weeks after each rain, favour Chironomus imicola which has a larval life span of as little as twelve days. This species relies on egg laying female flies to re-invade newly filled pools. In cases where the pool lasts for a shorter time than the minimum larval life span a mechanism is needed to survive dry periods in situ. Such pools are inhabited by larvae of Polypedilum vanderplanki and Dasyhelea thompsoni. Larvae of P. vanderplanki are poor at invading newly flooded pools and are also poor at competing with other species. However they are able to tolerate virtually a complete loss of body water and are therefore able to survive drought in the dry mud. They are consequently always the first there after refilling, a factor that provides them with sufficient advantage to eventually become established in very small pools. D. thompsoni larvae, not quite so good at surviving drought, but better at invading, occupy pools of intermediate size. They probably inhabit a larger number of pools than either of the other two species.A hazard associated with surviving dry phases in situ is vulverability to attack by terrestrial scavengers. However the activity of scavengers such as pheidolid ants, although conspicuous, is confined to periods when standing water has evaporated but the mud is still moist. Once dry, predation stops. Losses from this cause are therefore likely to be associated with periodic dry phases throughout the rainy season rather than with the dry season itself.

Characterization and heterogeneity of monoclonal antibodies directed to intestinal mucin derived from Crohn's disease small bowel.

A panel of 12 monoclonal antibodies were produced by immunization of Balb/c mice with small bowel epithelial cells obtained from a patient with active well-documented Crohn's disease. The clones were derived by screening with immunofluorescence microscopy; those with staining patterns suggestive of mucin directed epitopes were chosen for study. Several distinct patterns of staining reactivity were noted, including reagents with homogeneous, luminal, heterogeneous and peripheral goblet cell activity. In addition, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis revealed reactivity to high molecular weight mucin. The reactive antigen was resistant to proteinase digestion. No endoneuraminidase activity was detected; however, one neuraminidase sensitive sialic acid epitope was demonstrated. Confirmation of glycoprotein epitopes was accomplished by testing purified mucins from several areas of the gastrointestinal tract by ELISA. Finally, individual small bowel goblet cell heterogeneity was demonstrated by immunofluorescence, Western blotting, and antibody affinity chromatography. These data demonstrate both by morphology and specific binding of antibody affinity chromatography a significant degree of small bowel goblet cell mucin heterogeneity.

Contingency contracting with school problems.

Contingency contracting procedures used in managing problems with school-age children involved analyzing teacher and/or parental reports of behavior problem situations, isolating the most probable contingencies then in effect, the range of reinforcers presently available, and the ways in which they were obtained. The authors prepared written contracts delineating remediative changes in reinforcement contingencies. These contracts specified ways in which the child could obtain existing individualized reinforcers contingent upon approximations to desired appropriate behaviors chosen as incompatible with the referral problem behaviors. Contract procedures were administered by the natural contingency managers, parents and/or teachers, who kept daily records of contracted behaviors and reinforcers. These records were sent to the authors and provided feedback on the progress of the case. Initial results of this procedure have been sufficiently encouraging to warrant recommending an experimental analysis of contingency contracting as a clinical method.

Assessing clinical skills of medical students.

The clinical skills of sophomore medical students at the University of Arkansas are being assessed through the use of the Objective Structured Clinical Examination (OSCE). This exam was developed in order to better standardize the evaluation of practical clinical skills. The exam uses standardized patients, who are lay people trained to accurately and consistently portray a patient encounter. Faculty members at UAMS authored clinical cases for 20 patient encounters that test history taking, physical examination and communication skills. Each student interacts with the patient while being assessed in a standardized way, and then is given educational feedback by a faculty member. Students who do not pass the exam, undergo a remediation program prior to entering the junior year.