The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

Modulation of respiratory responses to carotid sinus nerve stimulation by brain hypoxia.

This study examines the effect of progressive isocapnic CO hypoxemia on respiratory afterdischarge and the phrenic neurogram response to supramaximal carotid sinus nerve (CSN) stimulation. Twelve anesthetized, vagotomized, peripherally chemodenervated, ventilated cats with blood pressure controlled were studied. During isocapnic hypoxemia, the amplitude of the phrenic neurogram was progressively depressed. In contrast, the increase in peak phrenic amplitude produced by CSN stimulation was unchanged, suggesting that the central respiratory response to CSN stimulation is unaffected by progressive hypoxemia. The time constant of respiratory afterdischarge (tau) was calculated from best-fit plots of phrenic amplitude vs. time after cessation of CSN stimulation. Under control conditions the value of tau was 57.7 +/- 3 (SE) s (n = 12). During progressive isocapnic hypoxemia, tau decreased as a linear function of arterial O2 content (CaO2) such that a 40% reduction of CaO2 resulted in a 48% reduction in tau. This reduction of respiratory afterdischarge may contribute to the genesis of periodic breathing during hypoxia.     

Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in lambda gt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.     

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation

The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation.     

Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver ( wv ) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation ( pcd ) destroys the vast majority of Purkinje cells. The staggerer ( sg ) and lurcher ( Lc ) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of G_D1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of G_TIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of G_D3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg , and Lc /+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that G_DT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for G_Tla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.     

Turnaround of Axoplasmic Transport of Selected Particle-Specific Enzymes at an Injury in Control and Diisopropylphosphorofluoridate-Treated Rats

: Reversal of direction (turnaround) of axonal transport of particle specific enzyme activities was studied at a ligature placed on rat sciatic nerve. In the principal experiment, the ligature remained on the nerve in vivo several hours, allowing enzyme activities (acetylcholinesterase, acid phosphatase, and monoamine oxidase) to accumulate immediately proximal to the tie. The nerve was then tied a second time, proximal to the first tie, and incubated in vitro for several more hours. Accumulation of enzyme activities just distal to the second tie was measured. This second accumulation, of activities traveling in the retrograde direction, was shown to be the result of turnaround in several ways. (1) The increase in activity distal to the second tie was equal to the decrease in activity proximal to the first. (2) The increase in enzyme activities distal to the second tie was greatly reduced when the accumulation proximal to the first tie was trapped by placing a third tie between the first and second ties. (3) It was shown that the activity that accumulated distal to the second tie could not have been in retrograde motion at the time of the first tie. (4) Accumulation distal to the second tie was not a function of the length of nerve segment included between the two ties. In contrast to the consistent occurrence of turnaround of orthograde flow, turnaround of retrograde flow could not be demonstrated. Turnaround transport was blocked by incubation in the cold and in the presence of NaCN or vinblastine. The turnaround process operated on all three enzymes studied, suggesting that it operates on lysosomes and mitochondria, as well as on the endoplasmic reticulum-like material bearing acetylcholinesterase. Evidence for the participation of the transport process in the renewal of AChE in the distal portions of the axon was obtained in experiments using diisopropylphosphorofluoridate and cycloheximide.