Aqueous two-phase extraction in bioseparations: an assessment.
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Biotechnology↗
Biomedical subjects
K D Cole
Publications and source records attributed to K D Cole.
Family dynamics and caregiver burden in home health care.
Planning an intervention involves the assessment of medical needs followed by an analysis of the family composition, dynamics, role allocations, coping mechanisms, mode of emotional expression, and the point in the family's developmental sequence. Therapeutic targets should be identified and prioritized. At that point, a behavioral probe or some specific recommendation that the family can easily follow should be made. A careful analysis of the response will indicate the ease with which more difficult change prescriptions can be introduced. Failure of the family to follow even the most simple suggestion may indicate that family therapy is needed. If the family is successful at carrying out the probe suggestion, however, the home health care team can proceed with greater confidence that their efforts will lead to improved patient care and healthy family functioning.
Aged↗
Purification of plasmid and high molecular mass DNA using PEG-salt two-phase extraction.
A method for the rapid preparation of DNA is described. The method utilizes a polymer (polyethylene glycol) and salt solution to form a two-phase system. A crude source of DNA is added to a phase-forming mixture, it is mixed and phase separation occurs. Under the appropriate conditions, the nucleic acids remain in the lower (salt-rich) phase, while the proteins, cellular debris and other constituents are in the upper phase (polymer-rich) or are precipitated at the interphase region. Incorporation of protein denaturants (detergents and chaotropes) stop the action of liberated nucleases in the sample. The nucleic acids are obtained in an intact state and in a form suitable for further manipulation, as shown by gel electrophoresis and DNA restriction digestion. This method describes the conditions of the two-phase systems that are important for the separation of nucleic acids and proteins. The important phase-forming conditions shown in this paper are pH, polymer molecular weight and concentration, salt type and concentration and the addition of detergents and chaotropic agents. With the use of these extraction conditions, proteins can be moved selectively from the lower to the upper phase. The paper describes a method for DNA isolation that is rapid, simple and economical.
DNA↗
Isolation of a genomic clone encoding the rat histone variant, H1d.
Mammals contain a family of five closely related H1 histone variants (H1a-e) as well as two less closely related forms, H10 and H1t. We have sequenced a rat genomic clone that encodes one of the standard H1 variants. An RNA transcript of the gene was made with bacteriophage SP6 RNA polymerase and translated in a cell-free system. The protein synthesized in vitro was identified as variant H1d by its electrophoretic mobility.
Amino Acid Sequence↗
The nucleotide sequence of a microvitellogenin encoding gene from the tobacco hornworm, Manduca sexta.
A microvitellogenin (mVg)-coding gene (mvg) has been isolated from a lambda phage library prepared from the tobacco hornworm, Manduca sexta. One of the lambda clones had a 15-kb insert and contained the entire mvg gene. A DNA fragment (3.0 kb) containing this mvg gene has been sequenced. Southern blot analysis showed that there may be more than one mvg gene in M. sexta. The putative transcriptional start point (tsp) for the cloned mvg was determined by primer extension analysis. This gene contains a single intron in the 5'-noncoding region. The 5'-flanking sequence was compared to the 5'-conserved regions of yolk polypeptide-encoding genes (yp) of Drosophila melanogaster. Two regions were found in the 5'-flanking sequence of the mvg gene that have 66% similarity to the D. melanogaster yp consensus sequence that is believed to be involved in gene expression controlled by ecdysteroids. Furthermore, the sequences flanking these two regions are also similar to the ecdysone-responsive elements found in several genes of D. melanogaster. In fact, preliminary experiments showed that mVg mRNA synthesis is induced by the 20-hydroxyecdysone. Four regions of the mvg gene resemble the upstream conserved regions of the two vitellogenin-encoding genes of the locust, Locusta migratoria. The nucleotide sequence of mvg has 70% similarity to the sequence of one or more of the 30-kDa hemolymph proteins of Bombyx mori. This indicates a very close evolutionary relationship between these proteins.
Amino Acid Sequence↗
cDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta.
Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.
Adipose Tissue↗
Nuclear transition protein 2 (TP2) of mammalian spermatids has a very basic carboxyl terminal domain.
Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.
Amino Acid Sequence↗
Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta.
The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.
Algorithms↗
Genes for chromosomal proteins expressed before and after meiosis.
Cloned gene sequences have been isolated for two testis-specific chromosomal proteins, one of which, histone (H1t), appears during meiosis, whereas the other, transition protein 1 (TP1), appears only during the later steps of spermatid development. Aspects of the regulation of each gene have been examined. In the case of H1t, analysis of its promoter region shows that it contains excellent matches to each of the four sequence homologies identified for the usual somatic H1 variants, so that the factor(s) that restrict H1t expression to spermatocytes remain a mystery. In the case of TP1, a cDNA clone allowed identification of its message by Northern blots as well as by in situ hybridization. The message appears postmeiotically in late round spermatids but is translationally repressed until the spermatid nucleus begins to condense.
Amino Acid Sequence↗
Geriatric assessment teams.
In geriatric care, a form of teamwork is the recommended modality because of the complex biopsychosocial needs of the patient. The goal of geriatric assessment programs is to establish an intensive assessment of older adults which requires the competencies of several coordinated disciplines. Not only do teams have the capacity to assess patients in much greater depth but also patients share different information with different providers. The composition of the team is dictated by the needs of the patient population in accordance with resources available. Next, one must identify a method of team practice in order for interactions to take place. The method of functioning determines what kind of team it is, ranging from independent functioning with minimal formal interfacing to interdependent activity interspersed with formal and informal interactions. In initiating a geriatric assessment program, one needs to determine which tasks demand interdisciplinary collaboration, which require interdisciplinary consultation, and which can be performed using a matrix or extended team model. In this model, the core team is supplemented by other disciplines as determined by the team, predicated on patient problems. Teams can profit from training, which can help with choosing an appropriate model, establishing a manual of procedure, and managing interactive issues and problems. This can occur early in the team's formation, or when a team takes on new members. The minimal level of team development would include establishing program goals, delineating professional responsibilities and roles, and implementing a system for exchanging and documenting information about patient plans. Saving input to share only in team meeting is inefficient, so health care teams need to recognize the importance of informal interchanges. It is still a matter of conjecture about what team works best with which patients under what circumstances or conditions. Multiple randomized clinical trials with teams will give us more information in this regard. In the meantime, organizers of geriatric assessment programs will have to make decisions based on clinical practice in the team development field and extrapolations from related health care team studies.
Aged↗
Geriatric assessment on a subacute hospital ward.
The authors collected data on all patients admitted to the Sepulveda Veterans Administration Geriatric Evaluation Unit (GEU) during its first 6 years of operation. Analysis of these data indicate several beneficial effects associated with this type of specialized geriatric care: improved diagnostic accuracy, reduced use of drugs, improved functional status, and improved placement location. The authors also analyzed additional data from a previously published, randomized controlled trial to compare better process of care between patients randomized to the GEU and those receiving usual services. During their initial hospitalizations, GEU patients received significantly more specialty evaluations than controls (4.9 versus 1.7, p less than .001), had longer lengths of stay (85.1 days versus 44.3 days, p less than .001), had more new diagnoses discovered (2.9 versus 0.6, p less than .001), and had more drugs discontinued from their regimens (4.6 versus 2.3 p less than .001). These process differences were probably related to the previously reported outcome differences: GEU patients were more likely than controls to show improvements in functional status, affect, placement location, use of institutional services, and survival.
Aged↗
Isolation of the gene for the testis-specific H1 histone variant H1t.
H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals. The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant. The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes. The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms. It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages. Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.
Amino Acid Sequence↗
Sequence of the amino terminal half of rat testis-specific histone variant H1t.
H1t is a testis-specific H1 histone variant that appears during the meiotic phase of spermatogenesis in mammals. The sequence of the first 108 residues of rat H1t have been determined and compared to boar H1t and also to a consensus sequence characteristic of standard somatic H1 variants. The two versions of H1t share a common pattern of divergence from the standard somatic consensus. For example, they both share many substitutions within the H1 globular region (residues 40-110), a portion of the molecule that is virtually invariant among the common somatic variants. Within the entire region sequenced, there are 28 shared locations where both forms of H1t differ from the somatic consensus. However, identical substitutions occur at only 15 of these sites, and each protein also differs from the consensus at five (boar) or ten (rat) additional locations that are not shared. These results establish that H1t from diverse sources shows a characteristic pattern of divergence from the sequence of standard somatic H1 proteins. However, it is also clear that there is great tolerance for species-specific variation within this H1 class.
Amino Acid Sequence↗
Understanding and interacting with older patients.
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Aging↗
Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases.
The mechanism that leads to an inhibition of enzyme activity in the presence of high concentrations of substrate was investigated with the two malate dehydrogenase isoenzymes obtained from pig heart. The inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of of oxalacelate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases from chicken hearts. The inhibition constants for oxalacetate are 2.0 mM with the mitochondrial enzyme and 4.5 mM with the cytoplasmic enzyme. Since the in vivo concentration of oxalacetate is reported to be about 10 micrometer, these data suggest that the substrate inhibition that is exhibited by the malate dehydrogenases may not be of any significance in vivo.
Animals↗