Defining ruminal and total-tract starch degradation for adult dairy cattle using in vivo data.

Literature was searched for studies performed in adult dairy cattle that simultaneously measured starch degradability in the rumen (RSDeg) and starch digestion in the total tract to compute postruminal starch digestion (PRSDig). Forty-one studies with 161 dietary treatments were used to form the data set. Of these diets, the major starch source was corn for 83 diets, small grain for 58 diets, and sorghum for 8 diets. Corn RSDeg was more variable than other sources. As measured in vivo across all starch sources, the percent RSDeg was influenced only by the amount of starch consumed, with the amount of degradation being approximately 75% at low starch intakes and decreasing to about 60% when 4 kg or more of starch were consumed. Small grain starch had greater RSDeg than corn or sorghum starch, which were approximately equal. The PRSDig of corn and small grain starches were approximately equal, but sorghum was about 15% less. Across all diets, models derived from the Cornell Net Carbohydrate Protein System predicted percentage of total-tract digestibility of starch very accurately, but overpredicted RSDeg and, as a result, underpredicted percent PRSDig. Calculation of RSDeg using a French model predicted the mean RSDeg with greater accuracy but less precisely. The relative differences in RSDeg percent among starch sources was correctly predicted by these models. A model using a revised rate of digestion as a way of combining effects of starch type and processing was developed, which predicted corn starch RSDeg and PRSDig with greater accuracy than nutrition models but only slightly better than using the mean observed degradation or the French calculation. Inaccuracies in prediction of RSDeg may be due mainly to processing effects and particle sizes, but these were not well reported in literature studies and were difficult to estimate. More accurate assessment of RSDeg and PRSDig will require better and more consistent reporting of grain processing. Based on this study, the French calculation is the most accurate of the models examined, although adjustments will be required to improve accuracy.

Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro.

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.

Specific regions of beta-globin RNA are resistant to nuclease digestion in RNA-protein complexes in chicken reticulocyte nuclei.

The interaction between beta-globin RNA and proteins in chicken reticulocyte nuclei was studied by determining the sequence of nuclease-resistant beta-globin RNA. Two types of nuclease-resistant RNAs were isolated for this study: endogenous nuclease-resistant RNA from 50S heterogeneous nuclear RNA-protein complexes and micrococcal nuclease-resistant nuclear RNA from whole nuclei. The nuclease-resistant regions were identified with the use of a RNA mapping method we recently developed (J.R. Patton and C.-B. Chae, J. Biol. Chem. 258:3991-3995, 1983). We found that beta-globin RNA is assembled into heterogeneous nuclear RNA-protein complexes in a specific manner. There are several regions of nuclease resistance in the first and third exons interrupted at regular intervals by sensitive regions. The second exon has only one nuclease-resistant region. The resistant regions range in size from 20 to 50 nucleotides. This organization may reflect a specific mode of assembly for heterogeneous nuclear RNA-protein complexes.

Reconstitution of the U1 small nuclear ribonucleoprotein particle.

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

Immunomodulatory and toxic effects of free and liposome-encapsulated tumor necrosis factor alpha in rats.

Tumor necrosis factor alpha has potent immunomodulatory and antitumor activity, but its therapeutic applications may be limited by its significant host toxicity. We showed that liposome-encapsulated recombinant human tumor necrosis factor alpha (rHuTNF-alpha) retained full anticellular activity in vitro. We then assessed the immunomodulatory and toxic effects of two different doses of i.v. free or liposome-encapsulated rHuTNF-alpha in normal rats. Both free and liposome-encapsulated rHuTNF-alpha significantly enhanced alveolar macrophage- and blood monocyte-mediated interleukin 1 release and tumor cell lysis, as well as natural killer cell cytotoxicity, when compared to buffer-treated controls. However, administration of rHuTNF-alpha in liposomes substantially reduced tumor necrosis factor alpha-mediated toxicity. Animals receiving liposome-encapsulated rHuTNF-alpha showed significantly less tissue injury, gastric retention, and circulating leukocyte shifts than animals receiving free rHuTNF-alpha. In addition, liposome-based delivery significantly increased lung and liver uptake of rHuTNF-alpha. Therefore, liposome-encapsulated rHuTNF-alpha retains immunomodulatory activity, significantly reduces toxic inflammatory effects, and may allow targeting of tumor necrosis factor alpha to selected organs after i.v. administration.

Pseudouridine formation in small nuclear RNAs.

Recent in vitro studies on the formation of pseudouridine (psi) in the spliceosomal small nuclear RNAs (snRNA) are reviewed. Multiple psi synthase activities, in some cases more that one per snRNA, are responsible for this modification of uridine. There is a requirement for Sm protein binding for the efficient formation of psi in U5 RNA but not for the modification of U2 RNA. The inhibition of psi formation by the incorporation of 5-fluorouridine in the snRNA is also reviewed.

Practical recommendations for using homework with students with learning disabilities.

Homework is a commonly used school practice. Its importance for students with learning disabilities has increased in recent years as these students have spent more instructional time in inclusive settings where homework is regularly given and as educational reforms have influenced the amount of homework being given. The first part of the article reviews selected issues that relate to using homework with students who have been identified as learning disabled. The major part of the article highlights effective (i.e., empirically validated) and recommended (i.e., suggested in the literature or determined by field-based reports) practices for using homework with this population.

Transition and students with learning disabilities: creating sound futures.

This article initiates a double issue that addresses a traditionally absent or rare piece in the system puzzle of preparing individuals with learning disabilities to meet the challenges of adulthood. Most professional efforts have focused on academic preparation for the 25 years or so that learning disabilities have been recognized. The rest of the person's adult adjustment (self-determination, life skills and community living, vocational preparation and employment, etc.) is presented within the framework of vertical and horizontal transitions, to organize the reader to consider all the options that youth must consider and prepare for. Individualized transition planning is discussed as a dynamic vehicle by which to empower students and families to utilize strengths, set and reach short-term and long-range goals, and include community variables in the process. Finally, an overview of the two issues describes their creation, their core common themes, and highlights of each article.

A life skills approach to mathematics instruction: preparing students with learning disabilities for the real-life math demands of adulthood.

Current mathematics instruction does not address the day-to-day needs of many students with learning disabilities. Although the vast majority of students with learning disabilities are not college bound, much of mathematics instruction provides college preparation. Too often, classes in mathematics ignore the skills needed in home and community and on the job. The present article examines the ways in which general mathematics instruction, focused on daily living skills, can easily be integrated into the classrooms of students with learning disabilities.

Mental retardation and learning disabilities: conceptual and applied issues.

The relationship between mental retardation and learning disabilities is clouded by conceptual issues and current practices in applied (i.e., educational and noneducational) settings. In this article, we initially discuss whether mental retardation can be considered a concomitant disability associated with learning disabilities or whether these two disabilities are mutually exclusive categories. Conceptual issues related to this question are then reviewed to provide a perspective for viewing these two traditional areas of exceptionality. Emerging areas of concern in term of definition, classification, etiology, and lifelong issues are addressed. Attention is then given to applied issues that have a direct effect on the lives of individuals with disabilities. Specific topics include educational curricula, instruction, inclusion, and adult services and supports.

In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells.

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.

Formation of pseudouridine in U5 small nuclear RNA.

The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There are three psi residues found in human U5 RNA, at positions 43, 46, and 53. Incubation of unmodified U5 RNA in reactions containing either S100 or NE supports psi formation at positions 43 and 46, which are found in a loop in the predicted secondary structure of U5 RNA. However, psi formation at position 53, which is found in a stem, is dependent on the presence of NE during the incubation. The order of extract addition does not have a significant effect on the formation of psi at position 53 as long as NE is present. The most efficient psi formation was observed with a combination of S100 and NE which allowed for efficient small nuclear ribonucleoprotein particle (snRNP) assembly and psi formation. When 9S and 20S U5 snRNPs were isolated by velocity sedimentation gradient centrifugation after incubation in the combined extracts, there was little difference in the psi levels at any of the positions for the two distinct particles. Mutations in the U5 RNA sequence do affect psi formation. U5 RNAs that have mutated Sm binding sites or are truncated prior to the Sm binding site have very low levels of psi formation at positions 43 and 46 and no detectable psi formation at position 53. A deletion of five nucleotides from 39 to 43 abolishes psi formation at positions 43 and 46, but the modification of position 53 is unaffected.

Ribonucleoprotein particle assembly and modification of U2 small nuclear RNA containing 5-fluorouridine.

An in vitro assembly/modification system was used to study the effect of 5-fluorouridine (5-FU) incorporation on the biosynthesis of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Labeled U2 RNAs were transcribed in vitro with 5-fluoro-UTP either partially supplementing or completely replacing UTP during synthesis. The resulting U2 RNAs have levels of 5-fluorouridine that range from 0 to 100% of the uridine content. When incubated in reactions containing extracts from HeLa cells, these 5-FU U2 RNAs are assembled into RNPs that are recognized by anti-Sm monoclonal antibody even when there is a complete replacement of uridine with 5-FU. However, when the in vitro assembled U2 snRNPs are subjected to buoyant density gradient centrifugation, the particles that contain 100% 5-FU are not resistant to salt dissociation. When the in vitro assembled U2 snRNPs were analyzed by velocity sedimentation gradient centrifugation, 5-FU incorporation correlated with a shift in the sedimentation rate of the particles. With 100% 5-FU incorporation, the peak of radioactivity shifted to approximately 15 S (control U2 RNA was at approximately 12 S). This peak from 5-FU U2 snRNPs was not resistant to dissociation on cesium sulfate gradients. The amount of pseudouridine (psi) found in the RNA from snRNP assembled in vitro on control and 5-FU-containing U2 RNAs was determined, and even at very low levels of 5-FU incorporation (5% replacement), the formation of psi was severely inhibited (36% of control). At higher levels of 5-FU incorporation, there was essentially no psi formed.

Multiple pseudouridine synthase activities for small nuclear RNAs.

The formation of pseudouridine (psi) in human U1, U2 and U5 small nuclear RNAs (snRNAs) was investigated using HeLa cell extracts. Unmodified snRNAs were synthesized in vitro and the extent of psi formation was determined after incubation in cell extracts. The formation of psi on labelled substrates was monitored in the presence of 5-fluorouracil (5-FU)-containing snRNAs as inhibitors of psi formation. The conversion of uridine to psi was inhibited only when the cognate 5-FU-containing inhibitor snRNA was included in the reaction. For example, 5-FU-containing U1 RNA inhibited psi formation in unmodified U1 RNA, but not in (unmodified) U2 or U5 RNAs. The results suggest that there are at least three activities that form psi in these snRNAs. The 5-FU-containing RNAs were stable during incubation in the cell extracts. A 12-fold molar excess of unlabelled U1 RNA did not inhibit psi formation on a labelled U1 RNA substrate, whereas a 3-fold molar excess of 5-FU-containing U1 RNA nearly abolished psi formation on the U1 substrate. The fact that 5-FU-containing snRNAs are potent inhibitors of psi formation in these pre-mRNA splicing cofactors raises the possibility that this is related to the cytotoxicity of fluoropyrimidines in cancer chemotherapy.

The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins.

The U1 small nuclear ribonucleoprotein (snRNP) particle, a cofactor in mRNA splicing, contains nine proteins, six of which are also present in other U snRNPs and three of which are specific to the U1 snRNP. Here we have used a reconstituted human U1 snRNP together with snRNP monoclonal antibodies to define the RNA binding sites of one of the U1 snRNP-specific proteins. When Sm monoclonal antibody (specific for the B', B, and D proteins of U snRNPs) was bound to U1 snRNPs prior to micrococcal nuclease digestion, the same approximately equal to 24 nucleotide fragment of U1 RNA (corresponding to nucleotides 120-143 and termed the "Sm domain") was protected as when no antibody was bound prior to digestion. In contrast, when RNP monoclonal antibody, which reacts with the U1 snRNP-specific Mr 70,000 protein, was bound, additional U1 RNA regions were protected against nuclease digestion. This phenomenon, which we term "antibody-mediated nuclease protection," was exploited to map the position of the Mr 70,000 protein to stem-loop I of U1 RNA. However, there were also sites of Mr 70,000 protein interaction with more 3'-ward regions of U1 RNA, particularly the Sm domain. This indicates that in the three-dimensional structure of the U1 snRNP, the RNP and Sm antigens are in contact with each other. The proximity of the Mr 70,000 protein's RNA binding site (stem-loop I) to the functionally important 5' end of U1 RNA suggests that this protein may be involved in the recognition of, or stabilization of base pairing with, pre-mRNA 5' splice sites.

Modification of human U4 RNA requires U6 RNA and multiple pseudouridine synthases.

Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions.