Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.     

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.     

Recombinant modified vaccinia virus Ankara-simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.     

Role of orlistat in the treatment of obese patients with type 2 diabetes. A 1-year randomized double-blind study

OBJECTIVE: Obesity is an important risk factor for type 2 diabetes. Weight loss in patients with type 2 diabetes is associated with improved glycemic control and reduced cardiovascular disease risk factors, but weight loss is notably difficult to achieve and sustain with caloric restriction and exercise. The purpose of this study was to assess the impact of treatment with orlistat, a pancreatic lipase inhibitor, on weight loss, glycemic control, and serum lipid levels in obese patients with type 2 diabetes on sulfonylurea medications. RESEARCH DESIGN AND METHODS: In a multicenter 57-week randomized double-blind placebo-controlled study, 120 mg orlistat or placebo was administered orally three times a day with a mildly hypocaloric diet to 391 obese men and women with type 2 diabetes who were aged > 18 years, had a BMI of 28-40 kg/m2, and were clinically stable on oral sulfonylureas. Changes in body weight, glycemic control, lipid levels, and drug tolerability were measured. RESULTS: After 1 year of treatment, the orlistat group lost 6.2 +/- 0.45% (mean +/- SEM) of initial body weight vs. 4.3 +/- 0.49% in the placebo group (P < 0.001). Twice as many patients receiving orlistat (49 vs. 23%) lost > or = 5% of initial body weight (P < 0.001). Orlistat treatment plus diet compared with placebo plus diet was associated with significant improvement in glycemic control, as reflected in decreases in HbA1c (P < 0.001) and fasting plasma glucose (P < 0.001) and in dosage reductions of oral sulfonylurea medication (P < 0.01). Orlistat therapy also resulted in significantly greater improvements than placebo in several lipid parameters, namely, greater reductions in total cholesterol, (P < 0.001), LDL cholesterol (P < 0.001), triglycerides (P < 0.05), apolipoprotein B (P < 0.001), and the LDL-to-HDL cholesterol ratio (P < 0.001). Mild to moderate and transient gastrointestinal events were reported with orlistat therapy, although their association with study withdrawal was low. Fat-soluble vitamin levels generally remained within the reference range, and vitamin supplementation was required in only a few patients. CONCLUSIONS: Orlistat is an effective treatment modality in obese patients with type 2 diabetes with respect to clinically meaningful weight loss and maintenance of weight loss, improved glycemic control, and improved lipid profile.     

Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E. coli cells and comparison of their functional activity]

Numerous data strongly suggest the involvement of cytokines and the matrix metalloproteinase collagenase (MMP-1) in the pathogenesis of periodontitis. Recently, we have demonstrated that, upon culturing under the influence of IL-1 alpha + EGF, a large amount of inactive procollagenase (MMP-1) is stored in the extracellular matrix of periosteal tissue. We now show that this endogenous reservoir of proenzyme can be operative after activation with plasmin and is able to induce a rapid and almost complete breakdown of the collagenous extracellular matrix. The level of collagen degradation following activation showed a strong correlation with the amount of proenzyme that was incorporated in the tissue. The highest level of degradation (70% of the total amount of collagenous proteins) was found with the IL-1 alpha + EGF-treated explants, followed by those treated with IL-1 alpha alone (35%). Explants cultured with EGF or in the absence of cytokines, containing only small amounts of procollagenase, showed little collagen breakdown following plasmin activation (7%). Inhibition of metalloproteinases by EDTA, or blockage of plasmin by PMSF, prevented the degradation in all explants irrespective of the amount of proenzyme present in the tissue. Our findings demonstrate that endogenous proenzyme stored in a native connective tissue matrix can be activated at a later time interval which results in a massive breakdown of the tissue. This study shows a possible pathway of collagenase-induced breakdown without recent de novo synthesis of the enzyme. Such a sequence may be operative in chronic inflammatory diseases, such as periodontitis, where production of procollagenase under the influence of cytokines spans a longer time period, whereas breakdown is often characterized by a cyclic behaviour.     

Karyological, biochemical, and physiological aspects of Callophysus macropterus (Siluriformes, Pimelodidae) from the Solim?es and Negro Rivers (central Amazon)

Karyological characteristics, i.e., diploid number, chromosome morphology and nucleolus organizer regions (NORs), biochemical characteristics, i.e., electrophoretic analysis of blood hemoglobin and the tissue enzymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), and phosphoglucose isomerase (PGI), and physiological characteristics, i.e., relative concentration of hemoglobin and intraerythrocytic concentrations of organic phosphates were analyzed for the species Callophysus macropterus collected from Marchantaria Island (white water system--Solim?es River) and Anavilhanas Archipelago (black water system--Negro River). Karyological and biochemical data did not reveal significant differences between specimens collected at the two sites. However, the relative distribution of hemoglobin bands I and III (I = 16.33 +/- 1.05 and III = 37.20 +/- 1.32 for Marchantaria specimens and I = 6.33 +/- 1.32 and III = 48.05 +/- 1.55 for Anavilhanas specimens) and levels of intraerythrocytic GTP (1.32 +/- 0.16 and 2.76 +/- 0.18 for Marchantaria and Anavilhanas specimens, respectively), but not ATP or total phosphate, were significantly different, indicating a physiological adaptation to the environmental conditions of these habitats. It is suggested that C. macropterus specimens from the two collecting sites belong to a single population, and that they adjusted some physiological characteristics to adapt to local environmental conditions.