The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

The distribution of keratin type intermediate filaments in human breast cancer. An immunohistological study

Antibodies to different intermediate filament proteins can be used to distinguish cells of epithelial, mesenchymal, muscle, glial and neuronal origin. Antibodies to prekeratin which characterize cells of epithelial origin, and antibodies to vimentin which recognize cells of mesenchymal origin have been used to study twenty cases of breast carcinoma (sixteen infiltrating ductal carcinomas and four infiltrating intraductal carcinomas), two cases of cystic breast disease, two fibroadenomas and one case of benign cystosarcoma phylloides. The prekeratin and vimentin were detected using specific antibodies to these proteins by immunofluorescence microscopy using alcohol fixed paraffin-embedded tissues. In eighteen out of the twenty carcinomas the tumor cells were strongly and specifically stained by antibodies to prekeratin. DIfferent tumors gave different patterns of prekeratin staining. In contrast, when the same specimens were tested with the vimentin antibody, the tumor cells were unstained, and instead only the usual strong staining to fibroblasts and blood vessels in the stroma was observed. In cystic breast disease, fibroadenomas, and benign cystosarcoma phylloides, cells of epithelial origin were strongly stained by the prekeratin but not by the vimentin antibody.     

Quantitative enzymatic, immunologic and histochemical studies of clinically relevant human kidney alterations using image analysis

Tissue sections of kidneys from 172 patients with various pathologic conditions, such as hydronephrosis, interstitial nephropathies, ischemia, chronic graft rejection and renal cancer, were evaluated by an image analysis technique. Structurally defined kidney alterations were monitored for enzymatic, immunologic and other histochemical changes. Indicator enzymes of the proximal tubule, alanine-aminopeptidase (AAP), alkaline phosphatase (AP), beta-glucoronidase (beta-Gl) and gamma-glutamyltranspeptidase (GGTP), were used as parameters for screening. Enzyme concentrations were found to be significantly decreased in kidney sections of patients with various renal diseases (AP less than 15%, AAP less than 55% and beta-Gl less than 60%) as compared to normal kidney tissues (100%). AAP concentration was measured quantitatively by specific immunofluorescence using an antienzyme antibody. Immunofluorescence of AAP was comparable to that of AAP calculated by the colorimetric technique (substrate: DL-alanine-beta-naphthylamide-HCl) and decreased to less than 50% in altered kidney tissues. Furthermore, kidney cancer (less than 20%) and kidney tissue adjacent to tumours (less than 65%) displayed significantly decreased levels of kidney marker enzyme activity. This study suggests that (1) the diseased kidney is characterized by a defined change in key enzymes of the cell surface and (2) renal cancer exhibits partial depletion of these constituents. Image analysis of the pattern of enzyme activity appears to be a useful tool in the analysis of renal pathology.     

An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain

A collection of antibodies specific to different intermediate filament proteins were applied to frozen sections of adult rat brains. The relative distribution of these proteins was then studied using double label immunofluorescence microscopy. Antibodies specific to each of the neurofilament "triplet" proteins (of approximate molecular weight 68 K, 145 K and 200 K) stained exclusively neuronal structures. The distribution of these three antigens was in general identical, except that certain neurofilament populations such as those in the dendrites and cell bodies of pyramidal cells of the hippocampus and cerebral cortex, contained relatively little if any 200 K protein. Some neurone populations, such as the granule cells of the cerebellar cortex, could not be visualized by neurofilament antibodies, indicating that neurofilaments may not be essential for function of all neurones in vitro. Antibodies to GFA and vimentin stained an entirely different population of processes, none of which stained with any of the neurofilament antibodies. Vimentin antibody stained sheath material around the brain, a monolayer of ependymal cell bodies lining the ventricles, fibrous material associated within the choroid plexus, the walls of blood vessels and capillaries, and the processes of cells in certain regions. GFA antibody stained a second layer of sheath material under the vimentin layer, and numerous processes visible throughout the brain. Some specific populations of GFA-positive processes proved to stain also with vimentin. These included the processes of Golgi "epithelial" cells (Bergmann glial fibres), those of certain astrocytes in bundles of myelinated fibers. In addition, some processes apparently derived from ependymal cells proved to stain for both vimentin and GFA, whilst other could only be reliably visualized by vimentin alone. These results are discussed in terms of the previously described morphological characteristics of the various cell types of the brain.     

Ultrastructure of the basiepithelial nerve plexus of the sea urchin,Centrostephanus longispinus

Summary In submandibular glands of rabbits both adrenergic and cholinergic axons are intimately associated with parenchymal cells of the intercalary ducts and the granular tubules, lying beneath the basement membrane and often in the space between the parenchymal cell and an associated myoepithelial cell. The submandibular acini receive a less intimate and less plentiful innervation by adrenergic and cholinergic axons which remain outside the basement membrane and are still associated with Schwann cells. Occasional axons of both adrenergic and cholinergic type occur beneath the basement membrane of submandibular striated ducts in intimate association with basal parts of the cells.In the parotid glands numerous adrenergic and cholinergic axons are found beneath the basement membrane of acini and intercalary ducts in intimate association with the cells.This work has been helped by the technical assistance of Mr. P.S.A. Rowley     

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Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Histone mRNAs contain blocked and methylated 5′ terminal sequences but lack methylated nucleosides at internal positions

Histone mRNA, labeled with ³²P or ³H-methionine during the S phase of partially synchronized HeLa cells, was isolated from the polyribosomes and purified as a “9S” component by sucrose gradient sedimentation. We identified two types of 5′ terminals, m⁷G(5′)pppN^mpN and m⁷G(5′)pppN^m-pN^mpN, in which the first methylated nucleoside is 7-methylguanosine, the second is either N⁶,2′-O-dimethyladenosine, 2′-O-methyladenosine, or 2′-O-methylguanosine, and the third is 2′-O-methyluridine, 2′-O-methylcytidine, or 2′-O-methyladenosine. Approximately 1.7% of the ³²P label was present in the 5′ terminal structures. Assuming a similar specific radioactivity for all phosphates, this percentage corresponds to an average of one terminal per 335 nucleotides. Histone mRNA differed from bulk polyadenylylated mRNA of HeLa cells in lacking significant amounts of 2′-O-methyluridine or 2′-O-methylcytidine in the second position of the 5′ terminal oligonucleotide and in lacking N⁶-methyladenosine residues at internal positions.