Cloning of a novel member (ARL5) of the ARF-family of Ras-related GTPases.

A novel ras-related GTPase with a unique structure was cloned by PCR-amplification with degenerate primers and screening of a rat fat cell cDNA library. The deduced amino acid sequence of the cDNA comprises all 6 GTP binding motifs which are conserved in Ras-related GTPases. The sequence is similar to that of ADP-ribosylation factors (ARF), and shows several structural features typical for the ARF-family. Because its closest relative is the GTPase ARL1 (49% identical amino acids, 54% identical nucleotides within the coding region), the protein was designated ARL5 (ARF-like protein 5). Low amounts of mRNA were found in most rat tissues examined (heart, skeletal muscle, fat, liver, kidney, lung, spleen, intestine, testis, and thymus) with highest levels in brain, intestine, and thymus.

In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin-diabetic rats.

The in vivo glucose uptake and the levels of two glucose transporter proteins (GLUT1 and GLUT4) were measured in heart and in various types of skeletal muscle from streptozotocin-diabetic rats. Diabetes (12-16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI), and the levels of GLUT1 and GLUT4 in heart by 75%, 60% and 70%, respectively. In diaphragm consisting of approximately equal amounts of type I (slow-contracting oxidative), IIa (fast-contracting oxidative) and IIb (fast-contracting glycolytic) fibers, GMI and GLUT4 levels were reduced by 60% and 40%, respectively, with no change in GLUT1 levels. In muscle consisting mainly of type I fibers (e.g., m. soleus), GMI and GLUT4 levels were reduced by 60% and 30%, respectively, whereas GLUT1 levels were unaltered. In mixed-type muscle consisting of type IIa and IIb fibers (e.g., m. plantaris and red part of m. gastrocnemius), GMI and GLUT1 levels were unchanged, whereas GLUT4 levels were decreased by 45%. In contrast, GMI was increased by 100% in type IIb fibers (e.g., the white part of m. gastrocnemius), probably reflecting the 4-fold increase in blood glucose levels, whereas GLUT4 levels were lowered by 55% with no change in GLUT1 levels. These data demonstrate a marked difference in the response of in vivo glucose uptake to long-term hypoinsulinemia between oxidative (type I) and glycolytic (type IIb) fibers. Furthermore, in contrast to the GLUT4, GLUT1 levels are regulated differentially in heart and skeletal muscle in response to streptozotocin-induced diabetes.

Evaluation of the reliability of 6 current anti-HIV-1/HIV-2 enzyme immunoassays.

The sensitivity for early detection of HIV antibodies and specificity of 6 anti-HIV-1/HIV-2 screening enzyme immunoassays (ELISAs) currently on the market were investigated by testing a panel of 249 well-characterized serum samples. The panel included sera from AIDS patients or children with congenital HIV infection, high-risk individuals and patients with conditions unrelated to AIDS. 'Tricky' sera (repeatedly positive results by ELISA and negative or indeterminate results by Western blot; n = 69) were also used in this evaluation along with 6 seroconversion panels. One second-generation assay (Biotest) and two third-generation assays (Abbott and Murex) showed the highest sensitivity for early detection of HIV-1 antibodies in seroconversion panels. A high specificity was achieved with the Cambridge Biotech (100%) and Ortho ELISA (99.4%). A relatively high rate of false-positive results was obtained with the Biotest (n = 10) and the Pasteur assays (n = 8) by testing 'tricky' sera and samples from high-risk individuals and from patients with other acute viral infections. In conclusion, it remains difficult to combine high specificity with an accurate detection of early seroconversion for anti-HIV-1/HIV-2 screening enzyme immunoassays.

Structure-activity relationship of covalently dimerized insulin derivatives: correlation of partial agonist efficacy with cross-linkage at lysine B29.

The effects of 7 covalently dimerized insulin derivatives on glucose transport in differentiated 3T3-L1 cells were investigated. Symmetric cross-linkage at lysine B29 with a bridge of 2 (oxalyl), 8 (suberoyl) or 12 (dodecanedioyl) carbon atoms produced derivatives with essentially unaltered receptor binding affinity but largely reduced intrinsic activity. Regardless of the chain length, these derivatives inhibited the effect of submaximal insulin concentrations. Insulin derivatives cross-linked at phenylalanine B1 or asymmetrically at B1/B29 were full agonists of the insulin receptor. When lysine B29 was cross-linked with the inactive desoctapeptide(B23-B30)insulin at phenylalanine B1, the intrinsic activity of the resulting dimer was lower than that of insulin, but higher than that of the symmetric B29-dimers. It is concluded that linkage at the B29-lysines, and not at the B1-phenylalanine, leads to partial agonism of dimerized insulin derivatives, regardless of the length of the crosslinker.

The effect of alcohol on emotional response to a social stressor.

Social drinkers were administered either an alcoholic, placebo or no-alcohol control beverage. Subjects were next informed that they were to give a self-disclosing speech about their body and physical appearance. Subjects' heart rate and videotapes of their facial expression were recorded during this instruction. Facial reactions to the stressor were analyzed using a system based on the Maximally Discriminative Facial Coding System (Izard, 1979). Subjects who were intoxicated showed significantly less negative emotion, as measured by the facial expression analysis, than those subjects consuming either the control or placebo beverage. We attribute this effect of alcohol to its actions on subjects' appraisal of anxiety-inducing information.

Inhibition of androgen receptor binding by natural and synthetic steroids in cultured human genital skin fibroblasts.

The ability of various natural and synthetic steroids (some of which are widely used in clinical practice) to compete with dihydrotestosterone receptor binding in human genital skin fibroblasts was studied. Binding was assessed in fibroblast monolayers after incubation for 1 h at 37 degrees C with 2 nM 3H-dihydrotestosterone in the presence or absence of increasing concentrations of the steroid to be tested. Inhibition constants (Ki) were determined as the concentration of competitor-required for 50% inhibition of 3H-dihydrotestosterone binding. In addition, relative binding activity (RBA) of each test compound was calculated. Each competitor was tested in at least two different cell strains. The concentrations of unlabeled methyltrienolone (a synthetic nonmetabolizable androgen) and dihydrotestosterone for 50% inhibition of 3H-dihydrotestosterone binding were in the same order of magnitude, namely, 2 nM (2.2 respectively, 2.4 nM), whereas the affinity of testosterone was approximately one-fifth that of dihydrotestosterone. Other potent competitors for dihydrotestosterone binding were three progestins (norgestrel, gestoden, and medroxyprogesterone acetate) which have Ki values similar to testosterone. An order of magnitude lower Ki values (around 10(-7) M) were found for the androgen 17 alpha-propylmesterolone, the antiandrogen cyproterone acetate, and the progestin norethisterone acetate. Binding affinities of all other steroids to the androgen receptor were markedly lower and showed the following order of potency: estrogens (estradiol, ethinyl estradiol, diethylstilbestrol) greater than glucocorticoids as well as aromatase inhibitors and potassium canrenoate.(ABSTRACT TRUNCATED AT 250 WORDS)

Approaching avoidance. A step essential to the understanding of craving.

Craving is only one component of the mental processes that influence drinking behavior. Alcohol-related cues (ARCs) can set in motion a dynamic competition between inclinations to approach drinking and inclinations to avoid drinking. Craving can thus be integrated into a comprehensive model of decision-making in which ambivalence or conflict is a key element. The relative strength of each component of the ARC reaction can fluctuate over time as well as in response to both subjective states and environmental circumstances. Simultaneously and independently evaluating these opposing responses puts clinicians in a better position to influence the relative weight that the patient assigns to the positive and negative outcomes of alcohol consumption.

True aneurysm of the pancreaticoduodenal artery: a case report and review of the literature.

A case of aneurysm of the pancreaticoduodenal artery is reported. Its differences from false aneurysms of the same artery are defined. The use of CT as a screening instrument and angiography as the definitive diagnostic tool is suggested to avoid undue delay in corrective treatment.

Cloning of two novel ADP-ribosylation factor-like proteins and characterization of their differential expression in 3T3-L1 cells.

A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.

Heterogeneity of insulin receptors in rat tissues as detected with the partial agonist B29,B29'-suberoyl-insulin.

Using the insulin receptor partial agonist B29,B29'-suberoyl-insulin, a covalently dimerized insulin derivative, we previously demonstrated a heterogeneity of signal transduction by insulin receptors in two cell systems. The present study was designed to characterize the heterogeneity of insulin receptors in different rat tissues with this agent. Binding of 125I-insulin to insulin receptors and its inhibition by B29,B29'-suberoyl-insulin or by unlabeled insulin were assayed in plasma membranes from brain, spleen, adipocytes, and liver. IC50 values of B29,B29'-suberoyl-insulin were different in all tissues investigated (brain < spleen < adipocytes < liver). In contrast, IC50 values of insulin were identical, with the exception of spleen membranes (spleen < brain = adipocytes = liver). Furthermore, the IC50 ratios (B29 dimer/insulin) were significantly different, ranging from 0.7 (brain) to 12.8 (liver). Solubilization and partial purification of insulin receptors failed to abolish the marked difference between brain and liver (IC50 ratios of 1.8 and 7.1, respectively). The apparent molecular masses of the alpha subunits of insulin receptors, as labeled with a photoreactive insulin derivative, appeared identical in liver and spleen but were significantly lower in adipocytes and brain (liver = spleen > adipocytes > brain). The tissue-specific expression of the known insulin receptor isoforms generated by alternative splicing (insulin receptor types A and B), as assessed by polymerase chain reaction amplification with oligonucleotide primers flanking exon 11, was not correlated with the differences in the IC50 values and ratios for insulin and B29,B29'-suberoyl-insulin. Furthermore, IC50 values of both insulin and the B29 dimer were 3-fold lower in membranes from Rat1 cells overexpressing insulin receptor type A, compared with membranes with insulin receptor type B; the IC50 ratios were identical. No additional alternative splicing of insulin receptor mRNA was found by polymerase chain reaction amplification and digestion with HaeIII and AluI of seven overlapping domains of the receptor alpha subunit. These data suggest a heterogeneity of insulin receptors in rat tissues that is unrelated to alternative splicing of the insulin receptor gene.

Two episodes of cytomegalovirus-associated colon perforation after heart transplantation with successful result.

A patient with end-stage heart disease was discharged from the hospital on postoperative day 7 after orthotopic heart transplantation. Three weeks from the day of operation, he developed sigmoid colon perforation, which required Hartmann's procedure, and 2 weeks later he had cecal disruption. A pathologic specimen showed underlying diverticular disease with associated cytomegalovirus colitis. Subsequently, the patient had multiple complications. Three months after colon perforation, the patient left the hospital, and now 1 year after transplantation he continues to do well. To the best of our knowledge, this is the first reported case of a patient who survived multiple colon perforations soon after heart transplantation.

ADP-ribosylation factor (ARF)-like 3, a new member of the ARF family of GTP-binding proteins cloned from human and rat tissues.

The human and rat homologues of a new member of the ADP-ribosylation factor (ARF) family of 21-kDa GTP-binding proteins, termed Arl3, were identified as an expressed sequence tag (human) and as a product of polymerase chain reaction amplification using degenerate probes derived from conserved sequences in members of the ARF family (rat). Alignments of the full-length open reading frames of the human and rat homologues revealed the encoded proteins to be over 97% identical to each other and 43% identical to human ARF1. Northern blots of mRNA from seven human tissues and four rat tissues revealed the presence of a ubiquitous band of about 1 kilobase in length that hybridized with the corresponding Arl3 probes. A number of human tumor cell lines expressed Arl3, as determined by immunoblotting with an Arl-specific antibody, raised against a peptide derived from the human Arl3 sequence. The level of Arl3 expressed in these cell lines was on the order of 0.01% of total cell protein. Purified recombinant human Arl3 was shown to bind guanine nucleotides but lacks ARF activity and intrinsic or ARF GTPase-activating protein-stimulated GTPase activity. In contrast to ARF proteins, the Arl3 protein has reduced dependence on phospholipids and magnesium for guanine nucleotide exchange. Thus, Arl3 is a ubiquitously expressed GTP-binding protein in the ARF family with distinctive biochemical properties consistent with its having unique, but unknown, role(s) in cell physiology.

Cardiac transplantation--first year experience in a community hospital: a three year follow-up.

This is a report of ten consecutive patients with end-stage cardiac disease treated with orthotopic cardiac transplantation in a community hospital, during the first year of its heart transplantation program. All patients were followed for a minimum of 33 months and a maximum of 45 months with 100% survival at two years and 90% at three years. All survivors are presently in N.Y.H.A. Class I or II. The entire group of patients received the same triple immunosuppressive therapy. The incidence of infection and rejection during the first three months post-transplantation was 0.3 and 0.6 episodes per patient respectively. Every patient developed some degree of deterioration in renal function and 80% of the patients now receive treatment for systemic hypertension. The in-hospital institution cost for the transplant admission varied from $25,084 to $74,164. To date, 30 patients have undergone heart transplantation in our program and 26 are long-term successes. This study again proves that renal insufficiency and hypertension remain the major side effects of Cyclosporine therapy. We further conclude from our experience that cardiac transplantation can be successfully and cost effectively performed in a community hospital even with a somewhat lower caseload.