Using nanoelectrospray ion mobility spectrometry (GEMMA) to determine the size and relative molecular mass of proteins and protein assemblies: a comparison with MALLS and QELS.

The determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool.

Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry.

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

Familial localized connective tissue nevus of the scalp with alopecia (report of a very unusual case).

Connective tissue nevi are benign hamartomas that usually appear as widespread, multiple, papular, skin lesions. They are subdivided into collagen, elastin, proteoglycans and mixed type, depending on their particular histopathologic features, and they often appear as a component of Buschke-Ollendorff Syndrome.

Comparison of biochemical polymorphisms and short tandem repeat (STR) DNA markers for paternity testing in rhesus monkeys (Macaca mulatta).

Genetic markers are indispensable for molecular and statistical genetic research involving nonhuman primates. Genetic markers must be used to ascertain parentage and to confirm the accuracy of pedigrees based solely on housing or demographic records; otherwise, the results of pedigree, linkage, or quantitative genetic analyses may be unreliable. Until recently, most genetic markers used in nonhuman primates were plasma proteins or isozyme polymorphisms, which were required in large numbers, because levels of genetic variation revealed by these markers were rather low. We compared the newer, PCR-amplified short tandem repeat markers (STRs) with a panel of classical biochemical polymorphic markers, for paternity determination among captive-bred rhesus monkeys. The STR markers exhibited an average genetic diversity of 64% and an expected paternity exclusion probability of 0.443. Both of these were greater than the average 54.5% genetic diversity and 0.298 exclusion probability exhibited by the biochemical markers. The STRs were much more efficient than the biochemical markers for parentage determination, since they required only half the amount of genetic typing data to resolve an average paternity case. Thus, the results of applying these two classes of genetic markers in paternity tests were somewhat different than expected on the basis of theoretical exclusion probabilities. These differences were probably due to inbreeding and other genetic differences among breeding colonies. Because they are more informative and provide rapid and efficient genetic data, STRs are now the method of choice for parentage determination and pedigree corroboration among nonhuman primates.

An unusual panniculitis appearing in the winter with good response to tetracycline.

Cold panniculitis is a form of physical panniculitis due to exposure of skin to severe cold. It usually appears on the cheeks of infants and children. It has also been reported on the thighs and buttocks of young females. Its clinical manifestations include red, cold, indurated plaques or nodules which appear one to three days after exposure to low temperatures and resolve spontaneously within several weeks without scarring. The histopathological picture shows a perivascular infiltrate of lymphoid and histiocytic cells at the dermal-subcutaneous junction in the early phase of the reaction (1). After 48 to 72 hours, a well developed panniculitis appears. We report an unusual case of an adult female patient with recurrent panniculitis on her legs appearing in the winter but without any preceding repeated or prolonged exposure to cold. She responded dramatically to oral tetracycline. This drug was successful as a prophylactic agent as well.

Disseminated and recurrent infundibular folliculitis (D.R.I.F.): report of a case successfully treated with isotretinoin.

Disseminated and recurrent infundibular folliculitis, henceforth referred to as D.R.I.F., is a very rare, puritic, follicular, benign disease of unknown etiology seen mostly in black males. It is often self-limited and usually unresponsive to local or systemic treatment. However, vitamin A, either alone or combined with vitamin E, is occasionally effective. We report a case of a patient with D.R.I.F. treated successfully with isotretinoin.

Analysis of the cDNA and encoded protein of the human testis-specific PGK-2 gene.

Because of their unique function, germ cells require unique gene products. Thus, although the glycolytic enzyme phosphoglycerate kinase (PGK) is required in all metabolically active cell types, there are two functional PGK genes in the mammalian genome, one, PGK-1, that is X-linked and ubiquitously expressed in all somatic tissues, and a second, PGK-2, that is autosomal and expressed only in spermatogenic cells. Expression of the PGK-2 gene may function solely to compensate for repressed expression of the PGK-1 gene due to X-chromosome inactivation in spermatocytes. Alternative y, the PGK-2 gene could encode an isozyme with unique characteristics that are beneficial to spermatozoa. We have isolated a cDNA of the human PGK-2 gene and used this as probe to demonstrate that transcription of this gene in spermatocytes and spermatids coincides with a period of repressed transcription of the X-linked PGK-1 gene during spermatogenesis in the human testis. We have also analyzed the amino acid sequence and protein characteristics of the PGK-2 isozyme deduced from this cDNA and compared them with that of the human PGK-1 isozyme to show that known structural and functional motifs are conserved in both proteins. Finally, we have examined the distribution of the PGK-1 and PGK-2 isozymes during spermatogenesis in the mouse to show that while the PGK-2 protein does not appear to possess any unique intracellular localization signal, it is more stable in vivo than the PGK-1 protein.

A primate model of monotypism in atherosclerotic lesions.

We have characterized the expression of allelic variants of X-linked glucose 6-phosphate dehydrogenase (G6PD) in aorta from homozygous, hemizygous, and heterozygous baboons (Papio hamadryas). Fibrous plaques from heterozygous baboons fed a high cholesterol, saturated fat diet contained distributions of G6PD allelic variants that differed from those of normal arterial wall and fatty streaks. The skewed allelic expression patterns in fibrous plaques of heterozygotes reflect decreased cellular heterogeneity in advanced vascular lesions. The tendency toward cellular monotypism in fibrous plaques is similar to that present in advanced human atherosclerotic lesions. Our results suggest that G6PD heterozygous baboons are a unique primate model for investigating the cellular origin of proliferating smooth muscle cells in atherosclerotic plaques.

X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons.

Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model.

Linkage heterogeneity between the C3 and LDLR and the APOA4 and APOA1 loci in baboons.

We detected linkage in baboons between loci for the third component of complement (C3) and the low-density lipoprotein receptor (LDLR), lod score = 5.53, and loci for apolipoprotein A1 (APOA1) and apolipoprotein AIV (APOA4), lod score = 14.59. We also found evidence for linkage heterogeneity among the baboon pedigrees between LDLR and C3 (P = 0.04) and between APOA1 and APOA4 (P = 0.01).

Adenine phosphoribosyl transferase polymorphism in baboons.

Two allelic isozymes of adenine phosphoribosyl transferase (APRT) were detected by starch gel electrophoresis of baboon hemolysates. Extensive family data verified autosomal codominant inheritance. The gene frequencies of five subspecies of baboons differed significantly. The activity of erythrocyte APRT is sufficiently high to enable the use of this enzyme as a sensitive marker for assessing chimerism in research involving bone marrow transplantation.

Linkage of plasminogen (PLG) and apolipoprotein(a) (LPA) in baboons.

Four allelic forms of serum plasminogen (PLG) were detected in baboons (Papio hamadryas Linneaus 1758) by isoelectric focusing and were determined to be inherited as autosomal codominant traits. Linkage analysis of data from 179 progeny and their parents revealed that PLG is tightly linked (lod score = 30.20) to the gene encoding apolipoprotein(a) (LPA), as in humans. No recombinant individuals were identified. This is the first linkage detected between PLG and LPA in any species other than humans and is the first genetic linkage identified in a nonhuman primate species by family studies.

Mannose-6-phosphate isomerase polymorphism in baboon erythrocytes.

Although erythrocytic mannose-6-phosphate isomerase (MPI) has been reported to be undetectable in mammals, we have demonstrated that sufficient activity is present in some species to enable its electrophoretic analysis. A survey of MPI from 2656 baboons revealed four allelic isozymes which segregated codominantly in pedigreed families. The gene frequencies differed significantly among five subspecies of baboons. However, the MPI*C allele had the highest frequency in all subspecies, ranging from 0.830 to 1.000. MPI phenotypes from erythrocytes and liver of the same individual were identical, indicating that the erythrocytic enzyme is specified by the same gene locus as the liver enzyme.

Biochemical genetic markers of squirrel monkeys and their use for pedigree validation.

Family data for 14 biochemical genetic markers fo squirrel monkeys (genus Saimiri) were derived from 73 pedigreed progeny and both parents of each, as well as from 16 additional progeny and one parent of each. The data for each marker and the phenotypic patterns were consistent with autosomal codominant inheritance. It was concluded from the genetic marker data that the pedigree records of seven progeny were incorrect. Retrospective investigations of colony records followed by typing of animals that might possibly have been a parent enabled five of the pedigree records to be corrected. Although five of the pedigree errors were cases of mistaken paternity, the other two apparently were the consequence of infant swapping between dams shortly after birth. Because squirrel monkeys exhibit a high degree of allomaternal behavior, infant swapping between dams may occur more frequently than in many other nonhuman primate species.

A photoaffinity derivative of colchicine: 6'-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine. Photolabeling and location of the colchicine-binding site on the alpha-subunit of tubulin.

A photoaffinity analog of colchicine, 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine, was synthesized by reacting deacetylcolchicine or [3H]deacetylcochicine with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Homogeneity of the photoaffinity analog was established by thin-layer chromatography and high-pressure liquid chromatography. The structure of the photoaffinity analog was determined by 1H and 13C NMR, infrared and ultraviolet-visible spectroscopies, and elemental analysis. Binding of 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine to bovine renal tubulin was measured by competition with [3H]colchicine. The value of the apparent Ki for the photoaffinity analog was 0.28 microM in the concentration range of 0.8-1.2 microM of the analog. A value of 0.50 microM for the apparent Kd was measured by the direct binding of the tritiated photoaffinity analog to tubulin. The analog is slightly more potent an inhibitor of microtubule formation than colchicine. The photoaffinity analog reacted with renal tubulin upon irradiation with a mercury lamp equipped with a 420-nm cutoff filter. Spectral and radiochemical analyses of the tubulin after photolysis and dialysis have demonstrated a stoichiometric incorporation of the photoaffinity analog in the alpha-subunit of the tubulin. Covalent labeling of tubulin with the photoaffinity analog decreases the extent of [3H]colchicine binding by more than 90%.

The effects of diet on growth and reproduction in gray short-tailed opossums (Monodelphis domestica).

In order to develop standard conditions for rearing the gray short-tailed opossum, Monodelphis domestica, as a potentially useful experimental laboratory animal, the effects of four different diets on growth and reproduction were assessed. One diet was a meat-based diet prepared in the laboratory. The other three diets were commercially produced fox foods designated Reproduction diet, Lactation diet, and Growing and Furring diet. All pairs of M. domestica fed the Reproduction diet produced at least one litter, but only two-thirds or fewer of the pairs fed any of the other three diets reproduced. There were no significant differences in the number of young born per litter or the number of young weaned per litter among the diets. Weight at weaning was significantly lower for individuals on the meat-based diet compared to those on the fox food diets. Young on the meat-based diet suffered 50% mortality within 6 weeks after weaning, whereas none of the animals fed the fox food diets died within the same 6-week period. Age-weight data were described using the Bertalanffy growth function. In terms of growth and overall reproductive performance, the fox food diets were clearly superior to the meat-based diet, and the Reproduction diet was judged to be the best of the fox food diets tested. Growth curves, from birth to 550 days of age, of individuals fed the Reproduction diet were developed and can be used as standards for the species under laboratory conditions. The maximal weights attained by animals fed the fox food diets were similar to the weights of the wild-caught founders of the laboratory population, indicating that the fox food diets provide adequate nutrition for normal growth. An additional observation was that females housed singly past the normal age of sexual maturity attained significantly lower adult weights than did females that were paired with males at 6 months of age.