Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Electromyography of brachial muscles in Pan troglodytes and Pongo pygmaeus

Electromyographic recordings were taken from all heads of the triceps brachii and biceps brachii muscles and from the anconeus, brachialis, and brachioradialis muscles in a chimpanzee and an orangutan as they stood still and walked quadrupedally on horizontal and inclined surfaces, engaged in suspensory behavior, reached overhead, and manipulated a variety of foods and artifacts. Like the gorilla (Tuttle and Basmajian, 1974a), the chimpanzee and orangutan possess special close-packed positioning mechanisms that allow the bulky muscles that cross their elbow joints to remain silent during quiet pendant suspension. We found no major myological features that would dramatically separate the arms of knuckle-walking African apes from those of the orangutan. With a few exceptions, which could as well be attributed to individual variation as to interspecific differences, the brachial muscles acted similarly during quadrupedal positional behaviors, irrespective of whether the hands of the subjects were knuckled (African apes), fisted (chimpanzee and orangutan), or placed in modified palmigrade postures (orangutan). Evolutionary transformations, from brachial and elbow complexes like those of Pongo to ones like Pan, or vice versa, would probably be achieved quite readily as the species changed its substrate preferences and positional habits.     

Structure-activity studies on high affinity NOP-active hexapeptides.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G-protein coupled receptor ORL1 (NOP), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiologic functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited number of high-affinity ligands to NOP, and particularly the lack of availability of useful specific antagonists. Herein, we describe the pharmacologic activity of a series of modified amino acid containing modifications of the hexapeptide Ac-RYYRWR-NH2, with high affinity for NOP. These compounds were tested for binding affinity using [3H]N/OFQ binding to human NOP in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS-binding in CHO cell membranes. These studies suggest that each Arg of the hexapeptide is required to maintain high-binding affinity. The peptide maintains high affinity if the Tyr2 or Tyr3 are modified, but at least one of these residues must maintain its hydroxyl group or there is a large decrease in intrinsic activity of the peptide.     

Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles.

We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophagy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4-fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient-deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vacuoles was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components.     

pH dependence and gene structure of inaA in Escherichia coli.

The weak-acid-inducible locus inaA in Escherichia coli was mapped to 48.6 min by P1 cotransduction of inaA Mud lac fusions and linked Tn10 insertions. The inaA1::lac fusion tested negative for phenotypes characteristic of mutations in the nearby locus ubiG. Sequence analysis of a fragment amplified by polymerase chain reaction located the inaA1::lac fusion joint within an open reading frame 311 nucleotides downstream of nrdB, transcribed in the opposite direction, encoding a 168-amino-acid polypeptide. Constitutive mutant strains identified on lactose MacConkey revealed a novel regulatory locus unlinked to inaA, which mapped at 34 min (designated inaR). Expression of inaA1::lac increased slightly with external acidification; the presence of benzoate, a membrane-permeant weak acid, greatly increased the acid effect. The expression at various combinations of benzoate and external pH correlated with the decrease in intracellular pH. The uncouplers salicylate and dinitrophenol also caused acid-dependent induction of inaA, but substantial induction was seen at external pH values higher than the internal pH; this effect cannot be caused by internal acidification. Nondissociating analogs of benzoate and salicylate, benzyl alcohol and salicyl alcohol, did not induce inaA. Expression of inaA was inversely related to growth temperature over the range of 30 to 45 degrees C. The inaA1::lac fusion was transferred to a strain defective for K+ uptake (kdpABC trkA trkD) in which pH homeostasis was shown to depend on the external K+ concentration. In this construct, inaA1::lac retained pH-dependent induction by benzoate but was not induced at low K+ concentrations. Induction of inaA appears to involve several factors in addition to internal pH. inaR may be related to the nearby locus marA/soxQ, which is inducible by acidic benzyl derivatives.     

Thiosulfate stimulation of microbial dark assimilation of carbon dioxide in shallow marine waters

The effect of thiosulfate on dark assimilation of carbon dioxide in shallow marine environments was investigated in order to explain the recent discovery of bacterial thiosulfate oxidation in aerobic, open ocean seawater. The results demonstrate that the potential exists for microbial thiosulfate oxidation to increase both dark assimilation of carbon dioxide and the utilization of organic compounds in the sea. Thiosulfate-stimulated microbial activity may be caused not only by chemoautotrophic sulfur bacteria, but also by heterotrophic species which oxidize thiosulfate to tetrathionate. Measurements of dark assimilation of carbon dioxide made at different incubation times indicate that great care must be taken both in experimental procedure and in interpretation of results obtained with the dark assimilation technique.     

Granular-cell tumor of the breast. A cytologic, immunohistochemical and ultrastructural study of two cases

Two primary granular-cell tumors of the breast are reported. Cytologically and histologically, these tumors had the classic features of small, eccentrically located nuclei and numerous periodic acid-Schiff-positive cytoplasmic granules. The characteristic cytologic features were best appreciated in touch imprints, not in frozen sections. Immunohistochemically, the tumors demonstrated diffuse cytoplasmic staining for S-100 protein but negative staining for myoglobin. The significance of these immunohistochemical staining characteristics, particularly in evaluating the possible histogenesis of these tumors, is discussed. The ultrastructural features of these two tumors are also presented and compared to findings reported by other investigators.     

Electromyography of pongid shoulder muscles III. Quadrupedal positional behavior

Electromyographic (EMG) recordings were taken from 14 shoulder muscles (or major parts of them) in a gorilla, a chimpanzee and an orangutan as they stood quadrupedally and tripedally, descended from elevated substrates, crutch-walked, and progressed quadrupedally on inclined and level substrates. In the African apes, low potentials commonly (but not always) occurred in the sternocostal pectoralis major, anterior deltoid, supraspinatus and subscapularis muscles during quadrupedal stance. The quadrupedal orangutan always exhibited low potentials in the pectoralis major muscle and EMG activity commonly occurred in her supraspinatus and subscapularis muscles. Quiescent tripedal stances were not accompanied by striking changes in EMG patterns from those which characterized quadrupedal stances. Per contra, eccentric loadings of the forelimb during descents from elevated substrates generally recruited notable EMG activity in the deltoid, supraspinatus and, to a lesser extent, infraspinatus muscles of the three pongid apes. The pectoralis major and caudal serratus anterior muscles were much more active in Pongo and Pan during these descents. Supportive segments of quadrupedal locomotive cycles were generally accompanied by EMG activity in the pectoralis major, intermediate and posterior deltoid and supraspinatus muscles. The intermediate and posterior deltoid muscles were characteristically active during pre-release of the hand and early swing phase. The cranial trapezius and supraspinatus muscles also may act during early swing phase. We conclude that the pectoralis major and perhaps the supraspinatus and subscapularis might serve regularly as postural muscles during static terrestrial quadrupedalism in pongid apes. The lack of dramatic differences between the EMG patterns exhibited during fist-walking versus knuckle-walking indicates that an evolutionary transformation from a shoulder complex like that of Pongo to ones like Pan or vice versa need not entail major changes in myological features.