Random mutagenesis of the cAMP chemoattractant receptor, cAR1, of Dictyostelium. Evidence for multiple states of activation

cAMP receptor 1 (cAR1) of Dictyostelium couples to the G protein G2 to mediate activation of adenylyl and guanylyl cyclases, chemotaxis, and cell aggregation. Other cAR1-dependent events, including receptor phosphorylation and influx of extracellular Ca2+, do not require G proteins. To further characterize signal transduction through cAR1, we performed random mutagenesis of the third intracellular loop (24 amino acids), since the corresponding region of other seven helix receptors has been implicated in the coupling to G proteins. Mutant receptors were expressed in car1(-) cells and were characterized for G protein-dependent and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third intracellular loop. Of the 21 positions where amino acid substitutions were observed, one or more replacements were found that retained full biological function. However, certain alterations resulted in receptors with reduced ability to bind cAMP and/or transduce signals. There were specific signal transduction mutants that could undergo cAMP-dependent cAR1 phosphorylation but were impaired either in coupling to G proteins, in G protein-independent Ca2+ influx, or in both pathways. In addition, there were general activation mutants that failed to restore aggregation to car1(-) cells and displayed severe defects in all signal transduction events, including the most robust response, cAMP-dependent cAR1 phosphorylation. Certain of these mutant phenotypes were obtained in a complementary study, where the entire region of cAR1 from the third to the seventh transmembrane helices was randomly mutagenized. Considered together, these studies indicate that the activation cycle of cAR1 may involve a number of distinct receptor intermediates. A model of G protein-dependent and -independent signal transduction through cAR1 is discussed.     

Effect of an enzymatic rinse on salivary levels of Streptococcus mutans and lactobacilli in periodontally treated patients

Root surface caries is prevalent in patients with both treated and untreated periodontal disease. The major etiologic factor has been identified as microbial plaque. In periodontally treated patients, significantly higher root caries prevalence and incidence have been found in patients with high levels of Streptococcus mutans and Lactobacilli in saliva. Reducing the levels of S. mutans and Lactobacilli in saliva may lower the risk of root caries development. The purpose of this investigation was to determine the effect of an oral enzymatic rinse on the salivary counts of S. mutans and Lactobacilli in periodontally treated patients. Fifteen adult subjects participated in a double-blind, cross-over designed clinical trial. Each subject had previously undergone comprehensive periodontal therapy and had been maintained on a regular program of supportive periodontal therapy. Paraffin-stimulated whole saliva was collected from each participant. Each subject was then randomly given either the enzymatic rinse product or a control rinse and instructed to rinse with one tablespoonful twice a day for 2 weeks, after which saliva samples were taken. After a washout period, salivary samples were again taken, and the subjects received the alternate rinse product. Two weeks later, final salivary samples were taken. The salivary samples were serially diluted and incubated aerobically on selective culture media. S. mutans and Lactobacilli were counted on the basis of colonial morphology. Pretreatment and posttreatment salivary counts of S. mutans and Lactobacilli were analyzed using the Wilcoxon matched-pairs signed-rank test at the 5% level of significance. Analysis of data revealed that neither the test nor the control rinse significantly lowered salivary counts of either species in the sample population.     

Transglutaminase II: a new class of GTP-binding protein with new biological functions

Tissue type transglutaminase (TGase II) is historically a member of the transglutaminase family, which covalently cross-links cellular proteins and polyamines. A recent new finding in the TGase II field is that the enzyme functions as a signal mediator from receptors to an effector in transmembrane signaling. This review will discuss the recent development of TGase II. This new signal transducer was termed Gh when initially discovered and was recently found to be TGase II. To help the reader understand the role of Gh as a signal mediator, the role of heterotrimeric G-proteins in hormone-mediated transmembrane signaling is briefly discussed. We have highlighted how Gh transmits the alpha 1-adrenoceptor signal to the phospholipase C-delta 1 and how Gh is activated and deactivated compared to the prototype of heterotrimeric G-proteins. Recent developments regarding the structure-function of Gh and other biological functions of Gh are discussed to facilitate understanding the impact of Gh in cells.     

The effects of colony-stimulating factor-1 on the number and ultrastructure of osteoclasts in toothless (tl) rats and osteopetrotic (op) mice

The role of colony-stimulating factor-1 (CSF-1 or M-CSF) in osteoclast development is illustrated by observations that administration of exogenous CSF-1 increases osteoclast number and improves the skeletal sclerosis of two osteopetrotic mutations, toothless (tl) in the rat and osteopetrotic (op) in the mouse. We examined the effects of CSF-1 treatment on the number and ultrastructure of osteoclasts in the tibial metaphysis of normal and mutant animals of both stocks to understand the similarities and differences between these two mutations. Osteoclasts from normal animals of both stocks were abundant and possessed the ultrastructural features of active cells. These included apical areas in contact with mineralized surfaces with tightly apposed clear zones, extensive ruffled borders, and a vacuolated cytoplasm with numerous mitochondria. In toothless rats osteoclasts were difficult to locate and those present had poorly defined ruffled borders, fewer cytoplasmic vacuoles, and a basal membrane with both smooth and ruffled areas. Large lipid accumulations were often found near tl osteoclasts. Osteoclasts in op mice were difficult to find, but more numerous than in tl rats. Unlike tl osteoclasts, those of op mice possessed very well developed ruffled borders, small clear zones, and large electron-dense cytoplasmic inclusions. These cells also had unusual basal membranes with both smooth and ruffled regions. CSF-1 treatment increased the number of osteoclasts in both mutant stocks, normalizing the numbers in op mice, but not tl rats. CSF-1 injections caused dramatic changes in the morphology of tl osteoclasts, including increased incidence and size of ruffled borders and cytoplasmic vacuolization. The growth factor had little effect on ruffled borders or clear zones in op mice. Interestingly, mutant osteoclasts of both stocks exhibited a ruffled basal membrane in response to CSF-1 treatment. This increase in membrane ruffling may reflect the ability of CSF-1 to promote rapid formation of osteoclasts from mononuclear precursors in a more permissive microenvironment. Our data indicate that CSF-1 is not required for the development of at least some osteoclasts. The differences in response to CSF-1 treatment which we report lead us to speculate that additional factors may be involved in osteoclastogenesis.     

Antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (IL-12) or other cytokines compared with exogenous IL-12

BACKGROUND: Numerous animal model studies have examined the ability of genetically engineered tumor cells to release cytokines and to elicit an immune memory against the parental tumor. Often only a single cytokine is studied, and few comparative studies have been conducted. PURPOSE: We evaluated the antitumor efficacy of adenocarcinoma cells engineered to release interleukin (IL)-12 in a mouse model system. The efficacy of this cytokine was compared with that of other cytokines released by engineered adenocarcinoma cells and that of exogenous IL-12 administered both locally and intraperitoneally. METHODS: BALB/cAnCr mice were inoculated with syngeneic parental mammary adenocarcinoma (TSA) cells in quantities sufficient to lead to tumors in all inoculated mice. TSA cells engineered to release IL-12 (TSA-IL12) were also injected into normal and selectively immunosuppressed BALB/cAnCr mice. Tumor incidence, growth, and rejection patterns were evaluated by the measurement of neoplastic masses and by the study of the histologic and ultrastructural features of the tumor site. The effects of local or intraperitoneal administration of recombinant IL-12 (rIL-12) on tumor-bearing animals were also studied. RESULTS: Most mice rejected TSA-IL12 cells through a CD8-positive, T-lymphocyte-dependent reaction associated with macrophage infiltration, vessel damage, and necrosis. The systemic immunity of mice that had rejected TSA-IL12 cells to a subsequent challenge with parental TSA cells was less efficient than that elicited by TSA cells engineered to release IL-4 or IL-10 but equivalent to that elicited by TSA cells engineered to release IL-2, IL-7, and interferon alfa. Compared with TSA cells engineered to produce other cytokines, TSA-IL12 cells were the most efficient in curing mice with established TSA tumors; injection of 0.1 million proliferating cells contralaterally to the tumor growth area cured five of 15 mice bearing 1-day-old tumors; injection of the same dose of proliferating cells into the tumor growth area cured two of 20 tumor-bearing mice. However, two 5-day courses with a nontoxic dose (0.1 microgram) of rIL-12 given intraperitoneally cured a similar proportion of these animals (six of 20). Only two of 20 mice with 7-day-old TSA tumors were cured by vaccination with proliferating TSA-IL12 cells, whereas 24 of 30 mice with such tumors were cured by intraperitoneal administration of rIL-12. CONCLUSIONS: TSA cells engineered to release IL-12 are rejected by most mice; the ensuing immune memory for TSA parental cells, however, was less efficient than that elicited by proliferating TSA cells engineered to release other cytokines (e.g., IL-4, IL-10, and possibly interferon gamma). The immune reaction elicited by TSA-IL12 cells was the most efficient in curing mice with established TSA tumors; notably though, the same or a better cure rate was obtained with rIL-12 given intraperitoneally.     

Using the Internet to engage youth in health promotion

A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.     

When do health care decisions discriminate against persons with disabilities?

Recent interpretations of laws prohibiting discrimination against persons with disabilities indicate that these laws will play a greater role in health care decision making than previously anticipated. This article employs lessons from other areas of antidiscrimination law to examine these developments and to provide a framework for making health care decisions that are consistent with these new legal interpretations. This article addresses decisions in individual cases, treatment policies adopted by health care providers, and coverage programs of third-party payers, both public and private.     

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin

A total of 663,533 colonies from 72 dairy and meat sources showed a detection rate of 0.2% for bacteriocin producers using direct plating techniques. A further 83,000 colonies from 40 fish and vegetable sources showed a detection rate of 3.4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus, Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.     

Autologous hapten-modified melanoma vaccine as postsurgical adjuvant treatment after resection of nodal metastases

PURPOSE: To determine whether treatment with an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP vaccine) is an effective postsurgical adjuvant treatment for melanoma patients with clinically evident nodal metastases. PATIENTS AND METHODS: Eligible patients had regional nodal metastases that were large enough (> or = 3 cm diameter) to prepare vaccine. Following standard lymphadenectomy, patients were treated with DNP vaccine on a monthly or weekly schedule. RESULTS: Of 62 patients with metastasis in a single lymph node bed (stage III), 36 are alive after a median follow-up time of 55 months (range, 29 to 76); the projected 5-year relapse-free and overall survival rates are 45% and 58%, respectively. Of 15 patients with metastases in two nodal sites, five are alive with a median follow-up time of 73 months. An unexpected finding was the significantly better survival of older patients; the projected 5-year survival of patients greater than 50 versus < or = 50 years was 71% and 47%, respectively (P = .011, log-rank test). The development of a positive delayed-type hypersensitivity (DTH) response to unmodified autologous melanoma cells was associated with significantly longer 5-year survival (71% v 49%; P = .031). Finally, the median survival time from date of first recurrence was significantly longer for patients whose subcutaneous recurrence exhibited an inflammatory response (> 19.4 v 5.9 months; P < .001). CONCLUSION: Postsurgical adjuvant therapy with autologous DNP-modified vaccine appears to produce survival rates that are markedly higher than have been reported with surgery alone. Moreover, this approach has some intriguing immunobiologic features that might provide insights into the human tumor-host relationship.