Quantitative Dot Blot Hybridization Research Articles

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

Virion stability is important for the circulative transmission of tomato yellow leaf curl sardinia virus by Bemisia tabaci, but virion access to salivary glands does not guarantee transmissibility.

The capsid protein (CP) of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV), family Geminiviridae, is indispensable for plant infection and vector transmission. A region between amino acids 129 and 152 is critical for virion assembly and insect transmissibility. Two previously described mutants, one with a double Q129P Q134H mutation (PNHD) and another with a further D152E change (PNHE), were found nontransmissible (NT). Another NT mutant with a single N130D change (QDQD) was retrieved from a new mutational analysis. In this study, these three NT mutants and the wild-type (wt) virus were compared in their relationships with the whitefly vector Bemisia tabaci and the nonvector Trialeurodes vaporariorum. Retention kinetics of NT mutants were analyzed by quantitative dot blot hybridization in whiteflies fed on infected plants. The QDQD mutant, whose virions appeared nongeminate following purification, was hardly detectable in either whitefly species at any sampling time. The PNHD mutant was acquired and circulated in both whitefly species for up to 10 days, like the wt virus, while PNHE circulated in B. tabaci only. Using immunogold labeling, both PNHD and PNHE CPs were detected in B. tabaci salivary glands (SGs) like the wt virus, while no labeling was found in any whitefly tissue with the QDQD mutant. Significant inhibition of transmission of the wt virus was observed after prior feeding of the insects on plants infected with the PNHE mutant, but not on plants infected with the other mutants. Virion stability and ability to cross the SG barrier are necessary for TYLCSV transmission, but interactions with molecular components inside the SGs are also critical for transmissibility.

Comparison of Microbial Community Compositions of Two Subglacial Environments Reveals a Possible Role for Microbes in Chemical Weathering Processes

Viable microbes have been detected beneath several geographically distant glaciers underlain by different lithologies, but comparisons of their microbial communities have not previously been made. This study compared the microbial community compositions of samples from two glaciers overlying differing bedrock. Bulk meltwater chemistry indicates that sulfide oxidation and carbonate dissolution account for 90% of the solute flux from Bench Glacier, Alaska, whereas gypsum/anhydrite and carbonate dissolution accounts for the majority of the flux from John Evans Glacier, Ellesmere Island, Nunavut, Canada. The microbial communities were examined using two techniques: clone libraries and dot blot hybridization of 16S rRNA genes. Two hundred twenty-seven clones containing amplified 16S rRNA genes were prepared from subglacial samples, and the gene sequences were analyzed phylogenetically. Although some phylogenetic groups, including the Betaproteobacteria, were abundant in clone libraries from both glaciers, other well-represented groups were found at only one glacier. Group-specific oligonucleotide probes were developed for two phylogenetic clusters that were of particular interest because of their abundance or inferred biochemical capabilities. These probes were used in quantitative dot blot hybridization assays with a range of samples from the two glaciers. In addition to shared phyla at both glaciers, each glacier also harbored a subglacial microbial population that correlated with the observed aqueous geochemistry. These results are consistent with the hypothesis that microbial activity is an important contributor to the solute flux from glaciers.

Attenuation of diurnal rhythms in plasma levels of melatonin and cortisol, and hypothalamic contents of vasotocin and isotocin mRNAs in pre-spawning chum salmon

In the present study, diurnal changes in plasma levels of melatonin and cortisol, and hypothalamic contents of neurohypophysial hormone mRNAs were examined in pre-spawning chum salmon, Oncorhynchus keta. From late November to early December, homing fish were captured at two sites along their migratory pathway on the Sanriku coast, Japan. Fish captured in the seawater (SW) environment were transferred to SW aquaria, and fish captured in the freshwater (FW) environment were to FW aquaria. They were maintained under natural photoperiod of approximately 10L:14D and sacrificed at 4-h interval through 24-h period. Plasma levels of melatonin were determined by radioimmunoassay, while cortisol levels were determined by enzyme immunoassay. Hypothalamic contents of vasotocin and isotocin mRNAs were determined by quantitative dot-blot hybridization assay. The melatonin levels showed weak nocturnal elevations in the SW and FW males, and FW females. The levels were maximal at 22:00 and minimal at 10:00 or 14:00, however the amplitudes were smaller than those reported in the previous studies using immature salmonids. The levels of vasotocin and isotocin mRNAs were higher in the males at all time points. The mRNA levels, however, did not show any diurnal variations in either of group. The same applied to plasma cortisol levels. These results indicate that the diurnal endocrine rhythms were attenuated in pre-spawning chum salmon, in contrast to the prominent diurnal rhythms in immature salmonids.

Colonic Bacterial Flora: Changing Understandings in the Molecular Age

The human intestinal microbiota is a complex bacterial consortium that is critical to normal health. The microflora is present at concentrations of 10(11)-10(12) cells/g of intestinal contents; the number of species present may exceed 500, although exact numbers remain to be defined, due in part to the fact that <30% of microorganisms are culturable with current microbiologic methods. Molecular tools based on 16S rDNA sequence similarities such as fluorescent in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), quantitative dot blot hybridization, restriction fragment length polymorphism (RFLP) and large scale 16S rDNA sequencing have helped to overcome limitations of conventional microbiological plating methods in studying the fecal microflora composition. However, these tools are just now beginning to be applied to understand the dynamics of this complex community, and its relationship to diet and human health. There is a need to understand both the limitations of the current data and the importance of moving forward with the best possible molecular and epidemiologic techniques as we deal with these critical questions.

Decreased Expression of Equine Herpesvirus-1 Early and Late Genes in the Placenta of Naturally Aborted Equine Fetuses

Intrauterine infection with equine herpesvirus-1 (EHV-1) has been considered to be the consequence of transplacental transmission of the virus following maternal cell-associated viraemia. In this study the state of EHV-1 gene expression in the placenta of seven naturally aborted equine fetuses was examined. Neither lesions nor viral antigens were detected in the placenta of the fetuses. The amount of infectious virus in the placentas was considerably lower than that in the fetal lungs, which showed pneumonia and typical herpesvirus inclusions. Quantitative dot blot hybridization with probes specific for immediate-early (IE), early (ICP0), and late (gD and gK) genes revealed that the placentas expressed the IE gene at a level comparable with that in the lungs; however, expression of the ICP0, gD and gK genes was significantly weaker in the placentas than in the lungs. In-situ hybridization demonstrated that both IE and gK RNAs were distributed mainly in the cytoplasm of trophoblasts. These results suggest that the low level of early and late gene transcription may be related to the limited production of viral progeny and the lack of immunoreactivity for viral antigen in trophoblasts infected with EHV-1.

Molecular Analysis of Intestinal Microbiota Composition to Evaluate the Effect of PEG and Lactulose Laxatives in Humans

Methods using bacteriological cultures allow only 30–50% of colonic bacterial populations to be detected. Our aim was to assess the effect of osmotic laxative administration on fecal bacteria during chronic idiopathic constipation, by a culture-independent molecular method. Forty-eight constipated patients were included in a randomized blinded trial and received either polyethylene glycol (PEG, n =23) or lactulose ( n= 25) for 28 days. Non-fermentable laxative (PEG) should not affect the bacterial groups, whereas fermentable (lactulose) could modulate them. The treatment dosage was 20 g per day for a 7-day period, then adjusted according to ef? cacy for 7 days and constant from 10 to 30 g per day for 14 days. Stools were collected at d0, d21 and d28, and analyzed by quantitative dot-blot hybridization using 16S ribosomal RNA-targeted oligonucleotide probes. We used a new Bifidobacterium group specific probe and five other probes to monitor the dominant groups of the intestinal bacteria: Bacteroides -Prevotella-Porphyromonas , Lactobacillus -Enterococcus- Streptococcus , enterobacteria: Clostridium coccoides and Clostridium leptum . Lactulose and PEG administration were associated with an increase of bifidobacterial rRNA index in 72% (18/25) and 17% (4/23) of cases, respectively. In the lactulose group, bifidobacterial rRNA index increased from [mean (SEM)] 1.1% (0.2%) at d0 (a), to 4.3% (1.2%) at d21 (b) and 4.5% (1.1%) at d28 (b), (a ≠ b, p=0.011). Lactulose treatment when administered at doses usually prescribed for constipation selectively stimulated bifidobacteria within the intestinal microbiota. The quantitative rRNA dot-blot hybridization method is an efficient tool to quantify intestinal bacteria, without the limitations of classical culture methods. Keywords: constipation, lactulose, quantitative dot-blot hybridization, Bifidobacterium probe, 16S rRNA.

Alendronate administration and skeletal response during chronic alcohol intake in the adolescent male rat.

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for alpha-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 +/- 13 mg/dl (E) and 203 +/- 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.

Molecular characterization of the human fecal flora using rRNA-targeted hybridization probes

Culture-based anaerobic techniques of microbial identification and enumeration are central to studies of the human fecal flora. Nevertheless, they are time-consuming, require fresh samples and are not suited to large epidemiological investigations. Moreover, comparison of cultureand microscopic-counts indicate that a significant fraction of the human fecal flora is not yet culturable. Molecular techniques should allow more accurate characterizations of extensive numbers of frozen fecal samples. The ai___m_m of this study was to assess rRNA-based quantitative dot-blot hybridization for the characterization of Bacteroides, Bifidobacterium, and Escherichia. coli populations of the human fecal flora in comparison with cultural counts. Methods: Total anaerobes, Bacteroides, Bifidobacterium and E. coli populations were enumerated from fresh fecal samples obtained from 10 healthy volunteers. Total RNA was extracted from the same fecal samples after storage at -20°C; hybridized with specific 32p-labeled oligonucleotide probes, and quantified by radio-imaging. Results are expressed as percent of total viable anaerobes for culture-counts, and as percent of total bacterial rRNA for dot-blot quantification. They are given as mean ± SE. Results: CoUnts of total viable anaerobes were 7.9 109 cellsdg feces (wet weight). Total Bacteroides represented 59.9± 16.7 % of total viable anaerobes (3.4 109/g) and 35.5 ± 4.5 % of bacterial rRNA, Bifidobacterium represented 10.6 ± 3.3 % of total viable anaerobes (7.4 10S/g) and 4.1 ± 0.7 % of bacterial rRNA, and Escherichia coli 5.4 ± 3.5 % of total viable anaerobes (1.4 10S/g) and 2.7 ± 0.7 % of bacterial rRNA. Conclusion: Relative rRNA proportions for the different bacterial groups were in good aggreement with accepted levels based on cultural counts. The two-fold higher proportion of all bacterial groups analyzed by culture compared to quantitative dot-blot was likely due to a systematic underestimation of counts of total viable anaerobes. The present work demonstrates that the rRNA approach bears great potential for the analysis of the human fecal flora and circumvents the limitations of culture-based techniques.

Investigation of c-myc and K-ras amplification in renal clear cell adenocarcinoma.

Tumour DNA samples isolated from 36 patients with renal clear cell carcinoma were investigated for c-myc and K-ras amplification, using a quantitative dot-blot hybridization. The characteristic clinical and histological parameters involved in the statistical analysis were age, sex, histological grade of the tumour, the TNM staging system, tumour size and weight, vascular invasion and the quality of life. The goal of the study was to estimate the prevalence as well as the prognostic value of the amplification of the oncogenes in question. Amplified c-myc (2.47-fold on the average) was found in three specimens (8.3%), showing slight correlation with intravasation (P > 0.05, n.s.). K-ras amplification (2.93-fold) detected in six tumours (16.6%) was shown to significantly correlate with both histological grade (2.2 vs. 1.8, P < 0.05) and tumour size (15 vs. 8 cm, P < 0.05). In cases with amplified K-ras also lymph node involvement was somewhat more frequent (P > 0.05, n.s.). No coamplification of these oncogenes was observed. The results of the study suggest that K-ras amplification may account for a more rapid progression of the disease.

Altered expression of γ2L and γ2S GABAA receptor subunits in the aging rat brain

Aging-related alterations in both protein and mRNA expression of gamma 2S and gamma 2L subunits of the GABAA receptors have been observed in several brain areas of Sprague-Dawley and Fischer 344 rats. Subunit-specific antibodies to gamma 2S and gamma 2L as well as a riboprobe to the large intracellular loop of gamma 2, which recognizes both gamma 2S and gamma 2L mRNAs, in conjunction with computerized image analysis were used for quantitative immunocytochemistry and in situ hybridization. In addition, specific oligonucleotide probes to gamma 2S or gamma 2L mRNA were used for quantitative dot blot hybridization. A large increase in the number of heavily immunostained neurons with the anti-gamma 2L antibody was detected in the cerebral cortex (115%) of old rats. However, only a small (but significant) aging-related increase in the density of gamma 2L immunostaining (7%) was observed throughout the cerebral cortex whereas no significant aging-related change in gamma 2L mRNA was detected in this brain region. Contrary to gamma 2L, the gamma 2S immunostaining did not show aging-related increased number of heavily immunostained neurons in cerebral cortex. Moreover, the density of gamma 2S immunostaining and the expression of gamma 2S mRNA were significantly decreased in the cerebral cortex (9-24%). Important aging-related changes were also found in the cerebellum of old rats where the expression of both gamma 2S and gamma 2L peptides was significantly decreased (24% and 23% respectively). This decrease in gamma 2 protein expression was accompanied by decreased expression of gamma 2S (16-38%) and gamma 2L (24%) mRNAs. Nevertheless, the most important decrease of gamma 2S (48%) and gamma 2L protein (20%) was revealed in the molecular layer of the cerebellum. In addition, the expression of gamma 2S protein was increased (14%) whereas the expression of gamma 2L was decreased (13%) in the granule cell layer. Therefore, the relative expression of gamma 2S protein in both layers was reversed in old animals. The observed aging-related changes in the expression of GABAA receptor subunits might lead to altered GABAA receptor/benzodiazepine receptor subunit composition.

Application of a low-light imaging device and chemiluminescent substrates for quantitative detection of viral DNA in hybridization reactions

In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.

Gene Expression After Uninephrectomy in Rat: Simultaneous Expression of Positive and Negative Growth Control Elements

Purpose Compensatory renal growth after contralateral nephrectomy consists largely of hypertrophy of the renal cortex. The signals initiating compensatory renal growth are as yet unknown. Materials and Methods Using a technique of quantitative dot-blot hybridization, we examined the expression of a number of genes after contralateral nephrectomy in the rat in an effort to establish a characteristic “fingerprint” which might shed light on the mechanism of compensatory renal growth. Results Negative growth control elements, including transcriptional repressors (WT-1, junB, myn, HNF-1, p53, RB, Dr1, gas2, gadd153) were induced 1.7 to 4.7-fold within 1 hour after uninephrectomy, as were positive growth control elements (egr-1, c-jun, cyclin C, cyclin E; 1.9-to 4.2-fold induction). The steady state mRNA levels for heat shock genes hsp70, hsp86 and hsp90 beta, as well as extracellular matrix genes fibronectin and collagen I (alpha 1 chain) were also increased 1 hour after uninephrectomy. In addition, 2 Na sup + -specific exchangers (NHE-1 and Na/Ca) were induced within 1 hour after uninephrectomy. Conclusions These results suggest the coordinate expression of positive and negative growth control elements early in this physiologic model of organ growth.

Strain evolution in Caenorhabditis elegans: Transposable elements as markers of interstrain evolutionary history

Evolutionary relationships across taxa can be deduced from sequence divergence of proteins, RNA, or DNA; sequences which diverge rapidly, such as those of mitochondrial genes, have been especially useful for comparisons of closely related species, and--within limits--of strains within a species. We have utilized the transposable element Tc1 as a polymorphic marker to evaluate the evolutionary relationships among nine Caenorhabditis elegans strains. For five low-Tc1-copy strains, we compared patterns of restriction fragments hybridizing to a cloned Tc1 probe. Twenty of the 40 Tc1 insertion sites thus characterized were common to all five strains, and so presumably preceded strain divergence; the 20 differential bands were used to construct a maximum-parsimony tree relating these strains. In four high-copy-number stocks (three wild-type strains and a subline), we determined occupancy of 35 individual Tc1 insertion sites by a polymerase chain reaction assay. Surprisingly, the high-copy strains share a common subset of these Tc1 insertions, and the chromosomal distribution of conserved Tc1 sites is "clustered" with respect to the other elements tested. These data imply a close evolutionary relationship among the high-copy strains, such that two of these strains appear to have been derived from the highest-copy-number lineage (represented by two stocks) through crossing with a low-Tc1 strain. Abundances of Tc1 elements were also estimated for the four high-copy-number stocks, at approximately 200-500 copies per haploid genome, by quantitative dot-blot hybridization relative to two low-copy strains. Annealing with 32P-labeled probes corresponding to full-length Tc1, an oligonucleotide within the Tc1 terminal inverted repeats, and an internal Tc1 oligonucleotide, gave essentially identical results--indicating that Tc1 termini exist in the genome primarily as components of full-length Tc1 elements. A composite evolutionary tree is proposed, based on the locations and numbers of Tc1 elements in these strains, which is consistent with a four-branch intraspecific tree deduced previously by maximum-parsimony analyses of mitochondrial sequence changes; it also serves to elucidate the evolutionary history of transposon mobility.

Investigation of c- myc oncogene amplification in colorectal cancer

Tumour DNA samples of 20 patients with colorectal carcinoma were tested for c- myc amplification, using a quantitative dot-blot hybridization. Statistical analysis involving clinical and histological parameters like degree of differentiation, Dukes' stage, TNM staging system, age, sex and severity of disease, was applied to estimate the prognostic value of c- myc amplification. The amplification of the investigated oncogene — 1.61-fold on the average — was found to significantly correlate with the presence of distant metastasis (corr. coeff.: 0.506, P < 0.05) and the severe course of the disease (corr. coeff.: 0.468, P < 0.05). This result supports the hypothesis that tumour cells with c- myc amplification represent a more malignant and aggressive phenotype. It is also worth noting that both c- myc amplification and formation of distant metastasis are late events in the progression of colorectal cancer, which accounts for the more severe course of the disease.

&lt;b&gt;AMPLIFICATION AND POINT-MUTATION ALTERATIONS OF ONCOGENES IN PATIENTS WITH CERVICAL CANCER-ASSOCIATED HUMAN &lt;/b&gt;&lt;b&gt;PAPILLOMAVIRUS &lt;/b&gt;

The infectivity of human papillomavirus (HPV) is closely linked to the characteristics of the epithelial lesions that they induce in the uterine cervix. Recent epidemiological studies have shown that not all HPV-infected patients necessarily develop carcinoma. It is therefore of importance to determine the molecular mechanism of tumorigenesis following HPV infection. In our previous study of amplification in the oncogenes c-myc and N-myc, amplification was observed in the c-myc but not in the N-myc gene. In the present study, we attempted to determine whether amplification occurs in other major oncogenes other than c-myc and N-myc, including c-erbB-2, N-ras and H-ras, using a quantitative dot-blot hybridization imaging plate method

Characterization of two variants of Panolis flammea multiple nucleocapsid nuclear polyhedrosis virus.

The baculovirus Panolis flammea multiple nucleocapsid nuclear polyhedrosis virus (PfMNPV) was originally isolated from a natural virus epizootic and shown to consist of a mixture of variants. Two subclasses of variants (PfMNPV A and B) were identified by Southern blot hybridization, their polyhedrin genes being located on different restriction fragments. The proportion of the A and B variants changed according to the larval host in which the virus was propagated; PfMNPV(B) predominated in P. flammea but PfMNPV(A) was predominant in Mamestra brassicae. Bioassays of the two pure virus variants in M. brassicae larvae have shown the LD50 values to be 4610 polyhedron inclusion bodies (pibs) for PfMNPV(A) and 5937 pibs for PfMNPV(B). Genomic DNA from the two variants was compared using restriction endonuclease analysis, and dot blot and Southern blot hybridization. Reciprocal quantitative dot blot hybridization analysis in 50% formamide showed PfMNPV(A) and PfMNPV(B) to be only distantly related to Autographa californica MNPV (less than 1%) and more closely related to M. brassicae MNPV (21 to 26%). The two PfMNPV variants exhibited a very high degree of identity to each other (nearly 100%) and therefore are very closely related. This was confirmed by physical mapping of the virus genomes. The nucleotide sequence of the polyhedrin gene of PfMNPV(B) was determined and compared with the published DNA sequences of other polyhedrin genes.

Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans.

A potentially lethal complication of trauma, malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.

Estrogen-Controlled Gene Expression: Induction of Two Estrogen-Responsive Genes in the Liver of Rainbow Trout (Salmo ǵairdneri)

Administration of 17β-estradiol to reproductively active male rainbow trout results in the induction of synthesis of two estrogen-responsive mRNA, vitellogenin (VG) and ULER2, to high levels. The complementary DNA (cDNA) of these two mRNA were prepared by molecular cloning. Two genomic DNA fragments generated by EcoRI digestion of trout genomic DNA showed hybridization to VG cDNA. A quantitative RNA dot blot hybridization method was adopted for the determination of levels of estrogen-responsive mRNA in livers of male trout after 17β-estradiol stimulation. This assay was used to determine the dose-response relationship and the time course of estrogen-responsive mRNA accumulation in juvenile fish.

Transient increases and individual variations in preprocholecystokinin gene expression in rat telencephalic regions during postnatal development: a quantitative comparison between preprocholecystokinin mRNA and preproenkephalin mRNA

Postnatal development of the tissue levels of mRNAs encoding preprocholecystokinin and preproenkephalin in rat telencephalic regions was analyzed by quantitative dot blot hybridization using synthetic RNAs as standards. Preprocholecystokinin mRNA levels per tissue weight showed distinct peaks in the frontal cortex and hippocampus on postnatal days 10 and 20, respectively, while preproenkephalin mRNA levels in these regions showed no apparent peaks. In the striatum, preproenkephalin mRNA abundance linearly increased 8-fold from day 5–43, while a transient increase in the preprocholecystokinin mRNA abundance between days 10 and 20 was noted. Furthermore, the striatal levels of preprocholecystokinin mRNA showed marked individual variations during the early period of postnatal development. These results suggest transient and variable increases in cell density and/or in gene expression of cholecystokinin-synthesizing cells in the developing telencephalon.