Reduced skin tumor development in cyclin D1-deficient mice highlights the oncogenic ras pathway in vivo

Cyclin D1 is part of a cell cycle control node consistently deregulated in most human cancers. However, studies with cyclin D1-null mice indicate that it is dispensable for normal mouse development as well as cell growth in culture. Here, we provide evidence that ras-mediated tumorigenesis depends on signaling pathways that act preferentially through cyclin D1. Cyclin D1 expression and the activity of its associated kinase are up-regulated in keratinocytes in response to oncogenic ras. Furthermore, cyclin D1 deficiency results in up to an 80% decrease in the development of squamous tumors generated through either grafting of retroviral ras-transduced keratinocytes, phorbol ester treatment of ras transgenic mice, or two-stage carcinogenesis.     

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

The biological properties of Aerococcus antagonists, representatives of human microbiocenoses

The paper deals with the data on biology of Aerococcus, a slightly studied group of microorganisms. Physiological-biochemical properties of Aerococcus are described, data of their distribution in nature are given. Peculiar attention is paid to the estimate of the role of Aerococcus in human microbiocenoses. As a result of the profound and all-round study of this group of microorganisms the authors have developed new bacterial drug "A-bakterin" based on the aerococcus strain. Data presented about the results of clinical tests of "A-bakterin" are presented, a possibility to use Aerococcus lysate in the elaboration of new drugs is discussed.     

The effect of combined antenatal vitamin K and phenobarbital therapy on umbilical blood coagulation studies in infants less than 34 weeks' gestation

OBJECTIVE: To determine if antenatal vitamin K and phenobarbital therapy affect coagulation studies in umbilical blood at birth, and to provide 95% reference ranges for umbilical blood coagulation parameters in premature gestations. METHODS: Patients at imminent risk for spontaneous or indicated premature delivery less than 34 weeks' gestation were randomized to receive either placebo or vitamin K and phenobarbital. Prothrombin time (PT), activated partial thromboplastin time (PTT), functional coagulation factors, and decarboxylated prothrombin assays were performed on umbilical blood specimens. Decarboxylated prothrombin, also known as "protein induced by vitamin K absence-factor II" or precursor prothrombin, is a sensitive marker for vitamin K deficiency. Standardized values of PT and PTT are reported in seconds and standardized values of factor assays in percentage of normal adult functional activity (mean +/- one standard deviation). RESULTS: Newborns in the placebo and treatment groups had similar umbilical blood PT (12.6 +/- 1.2 versus 12.7 +/- 1.4 seconds), PTT (48.8 +/- 13.4 versus 49.6 +/- 13.8 seconds), and functional activity of factor II (40.3 +/- 12.5 versus 42.0 +/- 12.1%), factor VII (67.0 +/- 20.9 versus 66.8 +/- 18.9%), factor IX (27.4 +/- 12.8 versus 25.8 +/- 8.9%), and factor X (47.0 +/- 12.8 versus 49.2 +/- 11.6%). Newborns in the treatment group were about half as likely as those in the placebo group to have detectable decarboxylated prothrombin levels in umbilical blood at birth (gestational age-adjusted odds ratio 0.47, 95% confidence interval 0.22-1.01; P = .05). CONCLUSIONS: Combined maternal therapy with vitamin K and phenobarbital before premature delivery does not affect umbilical blood PT, PTT, or functional activity of vitamin K-dependent coagulation factors II, VII, IX, and X. However, it is associated with the reduced presence of decarboxylated prothrombin in umbilical blood at birth. There is significant improvement in umbilical blood coagulation tests as gestational age advances from 24 to 34 weeks.     

Log-linear allometry of fetal craniofacial growth in Down's syndrome

Trisomy 21 develops as a result of nondisjunction of two homologous chromosomes during either the first or second meiotic division. One of the more important consequences of these genetic alterations is the predictable, although variable disturbance in the architecture of the craniofacial region [1]. Postnatal craniofacial morphology has been extensively studied in Down's syndrome (DS). However, little information is available on human prenatal development of the head and face in such patients. The time at which changes in craniofacial phenotype first emerge in Down's syndrome fetuses and at which physical growth begins to diverge from normal is unknown. To explore these questions, we compared prenatal craniofacial growth in 50 Down's syndrome fetuses with that of 555 fetuses judged to be "typical for body weight and age" using the method of log-linear allometry [2].     

Effect of insulin on farnesyltransferase. Specificity of insulin action and potentiation of nuclear effects of insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor

We have previously demonstrated that insulin activates farnesyltransferase (FTase) and augments the amounts of farnesylated p21 (Goalstone, M. L., and Draznin, B. (1996) J. Biol. Chem. 271, 27585-27589). We postulated that this aspect of insulin action might explain the "priming effect" of insulin on the cellular response to other growth factors. In the present study, we show the specificity of the effect of insulin on FTase. Insulin, but not insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF), stimulated the phosphorylation of the alpha-subunit of FTase and the amounts of farnesylated p21. Even though all four growth factors utilized the Ras pathway to stimulate DNA synthesis, only insulin used this pathway to influence FTase. Insulin failed to stimulate FTase in cells expressing the chimeric insulin/IGF-1 receptor and in cells derived from the insulin receptor knock-out animals. Insulin potentiated the effects of IGF-1, EGF, and PDGF on DNA synthesis in cells expressing the wild type insulin receptor, but this potentiation was inhibited in the presence of the FTase inhibitor, alpha-hydroxyfarnesylphosphonic acid. We conclude that the effect of insulin on FTase is specific, requires the presence of an intact insulin receptor, and serves as a conduit for the "priming" influence of insulin on the nuclear effects of other growth factors.     

Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle

The objective of the present study was to determine the duration of elevated concentrations of progesterone necessary to induce atresia of persistent ovarian follicles. Heifers were administered 25 mg of PGF2alpha on d 6 and 7 (d 0 = d of synchronized estrus) and a norgestomet implant from d 6 to 14. Ovaries were monitored by ultrasonography, and blood samples were collected on d 3, 5, 7, 9, 11, and 12 and daily from d 14 until ovulation. On d 12, heifers received either two progesterone-releasing intravaginal devices (PRID) for 6 h (6-h; n = 5), two PRID for 24 h (24-h; n = 5), or no treatment (CON; n = 5). Blood samples were collected at 15-min intervals from h -6 to 30 (PRID insertion = h 0) and analyzed for concentrations of LH. Characteristics of LH secretion were determined for consecutive 6-h periods (Period 0 to 5). Hourly blood samples, collected from h 0 to 29, were analyzed for concentrations of 17beta-estradiol (estradiol) and progesterone. The dominant ovarian follicles present on d 7 increased in size to 15.4+/-.3 mm on d 12 ("persistent follicle"). Following removal of the PRID and norgestomet implants, atresia of persistent follicles and ovulation of new follicles were induced in one of five and in four of five heifers in the 6-h and 24-h treatments, respectively. Persistent follicles ovulated after withdrawal of norgestomet in all other heifers. Concentrations of progesterone were increased from h 1 to 7 in the 6-h and h 1 to 26 in the 24-h treatment. Frequency of LH pulses was reduced (P < .05) during Periods 1 to 2 in the 6-h and Periods 1 to 5 in the 24-h treatment relative to the CON treatment. By h 10, concentrations of estradiol in the 6-h and 24-h treatments were lower (P < . 05) than in the CON treatment. This suppression continued through h 29 in the 24-h treatment (P < .05), whereas concentrations in the 6-h treatment were intermediate to those of the CON and 24-h treatments after h 14. Suppression of pulsatile LH release and estradiol secretion was evident with 6 and 24 h of treatment with progesterone, but only the 24-h treatment induced atresia of persistent follicles in a majority of the heifers.     

Comparison of the frequency and enjoyability of pleasant events in cocaine abusers vs. non-abusers using a standardized behavioral inventory

AIMS: To examine whether cocaine abusers differ from non-abusers in their frequency and enjoyability of engaging in various "pleasant events", in order to approximate the density of positive reinforcement experienced in their natural environment. DESIGN: Comparisons of cocaine abusers to normative data and matched controls. SETTING: An outpatient substance abuse treatment center in Burlington, Vermont, USA. PARTICIPANTS: Subjects included 100 individuals enrolled in outpatient treatment for cocaine abuse or dependence and 50 community volunteers without histories of drug abuse or other major psychiatric illness and matched to cocaine-dependent patients on age, sex and SES. MEASUREMENTS: Diagnostic assessments were based upon clinical interviews using the DSM-III-R checklist. The primary focus of this study was the Pleasant Events Schedule (PES), a self-rated behavioral inventory of the frequency and enjoyability of engaging in "pleasant" activities. Cocaine use history, treatment outcome and other relevant variables were also assessed. FINDINGS: Cocaine abusers reliably reported lower frequency of non-social, introverted, passive outdoor and mood-related activities than controls. These differences remained after controlling for demographic and life-style differences between groups, with the exception of mood-related activities. Perceived enjoyability of the activities did not differ across groups. Intravenous cocaine use and prior treatment for cocaine abuse predicted particularly low frequency of pleasant activities. Greater frequency of non-social activities predicted better treatment outcome. CONCLUSIONS: Drug abuse is associated with low density of certain types of non-drug reinforcement. Systematic increases in these activities may improve treatment outcome.     

Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin

Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.