Effects of the disruption of the HSP70-II gene on the growth, morphology, and virulence of Leishmania infantum promastigotes

The 70-kDa heat shock protein (HSP70) is highly conserved among both prokaryotes and eukaryotes and plays essential roles in diverse cellular functions not only under stress but also under normal conditions. In the protozoan Leishmania infantum, the causative agent of visceral leishmaniasis, HSP70 is encoded by two HSP70 genes. Here, we describe the phenotypic alterations of HSP70-II-deficient (Dhsp70-II) promastigotes. The absence of HSP70-II caused a major alteration in growth as the promastigotes reached stationary phase. In addition, aberrant forms were frequently observed in Dhsp70-II mutant cultures. An accumulation of cells in the G2/M phase in cultures of the Dhsp70-II mutant was determined by flow cytometry. Furthermore, Dhsp70-II promastigotes showed a limited capacity of multiplication within macrophages, even though attachment to and uptake by macrophages did not differ significantly from the wild-type. Moreover, Dhsp70-II was highly attenuated in BALB/c mouse experimental infections. In mutants re-expressing HSP70-II, the growth rate was restored, the normal morphology was recovered, and interactions with macrophages increased. However, promastigotes re-expressing HSP70-II did not recover their virulence. Overall, these data highlight the essential role played by HSP70-II expression in Leishmania virulence, pointing to this gene as a promising target for therapeutic interventions.     

Sequence conservation in the 3'-untranslated regions of neurone-specific enolase, lymphokine and protooncogene mRNAs

The C-terminal protein-coding and the entire 3'-untranslated regions of a cDNA corresponding to human neurone-specific enolase mRNA have been sequenced. The 3'-untranslated region is 892 bases long and shows a high degree of homology with the 3'-untranslated region of rat neurone-specific enolase mRNA. This sequence conservation is not seen in non-neuronal enolase mRNAs. Features of the conserved sequence include an A-rich region approx. 250 bases from the stop codon at a point corresponding to the polyadenylation signal site in non-neuronal enolase mRNA, and a repeating ATTT sequence. This unusual motif in eukaryotic mRNAs has previously been reported in the 3'-untranslated regions of lymphokine and protooncogene mRNAs.     

The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation

Background  

Exposure of Leishmania promastigotes to the temperature of their mammalian hosts results in the induction of a typical heat shock response. It has been suggested that heat shock proteins play an important role in parasite survival and differentiation.     

Expression of the HSP 70 gene family in rat hepatoma cell lines of different growth rates

We have studied the expression of different members of the HSP 70 gene family in MH1C1, FAO, and 3924A hepatoma cell lines, which possess different growth rates and show different levels of histone H3 gene expression. The cells have been subjected to mild (42 degrees C/1 h) or severe (45 degrees C/25 min) heat shock that causes a decrease in cell proliferation and histone H3 gene expression correlated to the severity of stress: previous mild heat shock protects against the effects of the subsequent severe exposure. All cell lines, irrespective of their growth rate, show a high constitutive expression of the HSC 73 gene, which is barely detectable in normal liver, and a good induction of the heat-inducible HSP 70 gene, which, however, seems to be induced less than in the normal tissue. The relative amount of grp 78 mRNA is high in all hepatoma cells lines, but only FAO cells maintain a significant expression of the albumin gene. The basic diversity in HSP 70 family gene expression between normal and tumors is still maintained in hepatoma cell lines, but the growth-related, quantitative differences among the transplantable hepatomas that we previously found in the animal (Bardella et al., Br. J. Cancer 55, 642-645, 1987; Cairo et al., Hepatology 9, 740-746, 1989), seem to be lost, or at least strongly blunted, in vitro.     

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 3'' untranslated regions of two related mRNAs contain an element highly repeated in the sea urchin genome.

Two closely related cDNA clones, pSpec1 and pSpec2, specifying two developmentally regulated tissue specific mRNAs from sea urchin embryos were used to probe a sea urchin genomic lambda library. Screening 10,000 phage by plaque hybridization yielded several hundred positive signals. With more stringent wash procedures, only two to three phage were positive. Three of these phage, one isolated by stringent wash procedures and two isolated by standard wash procedures were further investigated by restriction analysis, RNA gel blots, and DNA sequencing. The phage isolated by the stringent wash procedure appears to be a gene coding for the Specl mRNA. The other phage contain only partial homology to pSpec1 and pSpec2, 150 to 200 base pairs of the 3' untranslated region of the Spec1 and Spec2 mRNAs. It is concluded that the Spec1 and Spec2 mRNAs contain a highly repetitive element near their 3' end. The element is present at 2000 to 3000 copies per genome and may be transcribed at some sites other than those coding for the Spec1 and Spec2 genes. The possible function and evolutionary origin of the repetitive element is discussed.     

Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress

In order to study the effects of dietary emodin, high-dose vitamin C (Vc) and their combination on growth of Wuchang bream (Megalobrama amblycephala Y.) and its resistance to high temperature stress, 1200 healthy Wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. After feeding for 60 days, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to heat stress of 34 °C. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that before stress, compared with the control, the weight gain (WG) and specific growth rate (SGR), serum total protein (TP), lysozyme (LSZ), and alkaline phosphatase (ALP) levels, liver superoxide dismutase (SOD) activity and expression level of HSP70 mRNA significantly increased in emodin and Vc groups while feed conversion rate (FCR), serum cortisol (COR), triglyceride (TG) and liver malondialdehyde (MDA) contents decreased (P < 0.05); liver catalase (CAT) activity also significantly increased in emodin group (P < 0.05). Although serum TP, LSZ, and liver HSP70 mRNA levels significantly increased and liver MDA level decreased in combination group (P < 0.05), no synergism was observed. After heat stress, compared with the control, the serum TP, LSZ, ALP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P < 0.05) while the difference between the combination and control groups was not significant (P > 0.05). Thus, the basal diet supplemented with 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, reduce FCR, increase non-specific immunity of fish, antioxidant capacity, and two HSP70s mRNA expression levels, and enhance resistance to heat stress in fish. However, the combination of emodin and high-dose Vc showed no better effect.     

Multiple red cell ferritin mRNAs, which code for an abundant protein in the embryonic cell type, analyzed by cDNA sequence and by primer extension of the 5'-untranslated regions

Ferritin maintains iron in a bioavailable, nontoxic form for vertebrates and invertebrates, higher plants, fungi, and bacteria; the protein is formed from two classes of subunits (H and L) in ratios which vary in different cell types. Ferritin may be an abundant, differentiation-specific protein or a "housekeeping" protein. The red cells of embryos are specialized for iron storage and have abundant ferritin; iron regulates the synthesis of ferritin in such cells translationally by recruitment of stored, ferritin mRNA and by translational competition. To characterize mRNA regulated in such a manner, we prepared cDNA from reticulocytes of bullfrog tadpoles, a readily available source of embryonic red cells; moreover, no protein sequence information was available for nonmammalian ferritin. An almost full-length (817 base pairs) cDNA (pJD5F12) was isolated and sequenced, the 5' end was analyzed by primer extension, and the cloned DNA was used as a hybridization probe. We have shown that ferritin mRNA is stored in the cytoplasm and that the 5' end of the mRNA is heterogeneous. The 5'-untranslated region of ferritin mRNA consisted of 143 nucleotides in the major (65%) species and 146 or 152 in the minor species (approximately 17% each). (Heterogeneity is characteristic of some other abundant mRNAs, e.g. globin, which is also translationally regulated.) Since excess iron had no detectable effect on the heterogeneity of the 5' end of ferritin mRNA, the feature is more likely associated with mRNA abundance and/or cell specialization than translational control. In the bullfrog, as in humans and rats, ferritin is encoded by multiple genomic sequences (four to eight) which specify proteins of considerable homology. For example, 75 of the 81 amino acids present in all mammalian ferritins sequenced are also present in the frog; the overall homology between frogs and humans or rats is 59-66%. Ferritin H and L subunits in humans are distinct (overall homology 56%) and appear to have diverged from a common precursor relatively recently. In contrast, ferritin H and L subunits have high homology in tadpole red cells, determined by hybrid select translation, which suggests that bullfrog red cell ferritin may be close to the primordial sequence.     

Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle

The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.