Electron Microscopy of Herpes Simplex Virus: I. Entry

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.     

Pathways of glycosphingolipid biosynthesis in SW13 cells in the presence and absence of vimentin intermediate filaments

We reported previously that the incorporation of sugars intoglycosphingolipids (GSL) is diminished in SW13 cells that lacka vimentin intermediate filament (IF) network (vim–) comparedto vim+ cells. To further analyze the nature of this abnormality,we double-labeled cells with ³H-serine and ^l4C-sugars. Therewas no difference between vim+ and vim– cells in the incorporationof serine into GSL, although the usual difference in sugar incorporationwas observed. This indicated that the defect in vim– cellswas not in the incorporation of sugars into ceramide synthesizedde novo by acylation of sphinganine (pathway 1). Sugars canalso be incorporated into ceramide synthesized from sphingosinethat is derived from catabolism of sphin-golipids (pathway 2),and into GSL that recycle through the Golgi apparatus from endosomes(pathway 3). The amount of galactose and glucosamine incorporatedinto GSL in these three pathways was analyzed by the use oftwo inhibitors of sphingolipid biosynthesis. ß-Chloroala-nineinhibits the de novo synthesis of sphinganine (pathway 1), andfumonisin Bl inhibits the acylation of sphinganine and sphingosine(pathways 1 and 2). We were surprised to observe that in bothvim+ and vim– cells only 20–40% of sugar incorporationinto GSL took place in pathway 1, and 60–80% of sugarincorporation took place in the recycling pathways. Moreover,in contrast to larger GSL, GlcCer was not synthesized in pathway3. Our observations indicate that vimentin IF facilitate therecycling of GSL and sphingosine, and that the differences betweenvim+ and vim– cells are predominantly in pathways 2 and3. Furthermore, although it is generally believed that virtuallyall GSL are synthesized in the de novo pathway, these data indicatethat the recyling pathways predominate in the incorporationof sugars into GSL in SW13 cells. glycosphingolipid biosynthesis intermediate filaments SW13 cells sphingolipid recycling vimentin     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A(2)

Small, dense, electronegative low density lipoprotein [LDL(-)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(-) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(-) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(-) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(-). In contrast, lipoprotein-associated phospholipase A(2) (LpPLA(2)) is highly enriched only in small, dense LDL(-). The association of LpPLA(2) with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core.     

Direct Measurement of the Structure of Reconstituted High-Density Lipoproteins by Cryo-EM

Early forms of high-density lipoproteins (HDL), nascent HDL, are formed by the interaction of apolipoprotein AI with macrophage and hepatic ATP-binding cassette transporter member 1. Various plasma activities convert nascent to mature HDL, comprising phosphatidylcholine (PC) and cholesterol, which are selectively removed by hepatic receptors. This process is important in reducing the cholesterol burden of arterial wall macrophages, an important cell type in all stages of atherosclerosis. Interaction of apolipoprotein AI with dimyristoyl (DM)PC forms reconstituted (r)HDL, which is a good model of nascent HDL. rHDL have been used as an antiathersclerosis therapy that enhances reverse cholesterol transport in humans and animal models. Thus, identification of the structure of rHDL would inform about that of nascent HDL and how rHDL improves reverse cholesterol transport in an atheroprotective way. Early studies of rHDL suggested a discoidal structure, which included pairs of antiparallel helices of apolipoprotein AI circumscribing a phospholipid bilayer. Another rHDL model based on small angle neutron scattering supported a double superhelical structure. Herein, we report a cryo-electron microscopy-based model of a large rHDL formed spontaneously from apolipoprotein AI, cholesterol, and excess DMPC and isolated to near homogeneity. After reconstruction we obtained an rHDL structure comprising DMPC, cholesterol, and apolipoprotein AI (423:74:1 mol/mol) forming a discoidal particle 360 Å in diameter and 45 Å thick; these dimensions are consistent with the stoichiometry of the particles. Given that cryo-electron microscopy directly observes projections of individual rHDL particles in different orientations, we can unambiguously state that rHDL particles are protein bounded discoidal bilayers.     

2-Hydroxylation of estrogens in the brain participates in the initiation of the preovulatory LH surge in the rat

Estradiol-2-hydroxylase, the enzyme responsible for the conversion of estrogens to catechol estrogens was measured in the brain of female rats at specific stages of the estrus cycle. Radiometric measurements of the enzyme activity in microsomal, mitochondrial, and synaptosomal fractions of the brain revealed a sharp increased in activity at proestrus just prior to the preovulatory LH surge. The enzyme activity declined to lower levels at diestrus and metestrus. No comparable fluctuations were noted in the liver enzyme. These changes in brain enzyme activity in conjunction with demonstrated positive feedback of exogenous catechol estrogens on pituitary LH release, suggest that a rise in endogenous catechol estrogen formation in the brain may be responsible for the physiological induction of the preovulatory LH surge.     

Mitochondrial DNA origins of the Latvian clefting population

Latvia has one of the highest prevalence of isolated cleft lip with or without cleft palate (CL/P) in Europe. To clarify the genetic origins of the Latvian cleft population and establish a method for genetic mapping, mitochondrial DNA variation was studied in a population affected with clefting. One-hundred and seven subjects and 351 samples from unrelated healthy volunteers representing four anthropologically, archaeologically and ethno-linguistically different regions of Latvia were selected. The case group showed a higher frequency of haplogroups U4 (p=0.02) and U5 (p=0.0003) than in non-U haplogroups. We hypothesize that U4 and U5 mtDNA haplotype carriers may also carry susceptibility genes for clefts. Future studies will take into consideration these definitions based on mtDNA haplotypes when analyzing genetic variations and their possible contribution to CL/P susceptibility.     

HIV-1 Specific Antibody Titers and Neutralization among Chronically Infected Patients on Long-Term Suppressive Antiretroviral Therapy (ART): A Cross-Sectional Study

The majority of potent and broadly neutralizing antibodies against HIV-1 have been isolated from untreated patients with acute or chronic infection. To assess the extent of HIV-1 specific antibody response and neutralization after many years of virologic suppression from potent combination ART, we examined antibody binding titers and neutralization of 51 patients with chronic HIV-1 infection on suppressive ART for at least three years. In this cross-sectional analysis, we found high antibody titers against gp120, gp41, and the membrane proximal external region (MPER) in 59%, 43%, and 27% of patients, respectively. We observed significantly higher endpoint binding titers for gp120 and gp41 for patients with >10 compared to ≤10 years of detectable HIV RNA. Additionally, we observed higher median gp120 and gp41 antibody titers in patients with HIV RNA <50 copies/mL for ≤5 years. 22% of patients neutralized a HIV-1 primary isolate (HIV-1_JR-FL) and 8% neutralized a HIV-2/HIV-1 MPER chimera. Significantly greater HIV-1_JR-FL neutralization was found among patients with >10 years of detectable HIV RNA (8/20 [40.0%] versus 3/31 [9.7%] for ≤10 years, p = 0.02) and a trend toward greater neutralization in patients with ≤5 years of HIV RNA <50 copies/mL (7/20 [35.0%] versus 4/31 [12.9%] for >5 years, p = 0.08). All patients with neutralizing activity mediated successful phagocytosis of VLPs by THP-1 cells after antibody opsonization. Our findings of highly specific antibodies to several structural epitopes of HIV-1 with antibody effector functions and neutralizing activity after long-term suppressive ART, suggest continuous antigenic stimulation and evolution of HIV-specific antibody response occurs before and after suppression with ART. These patients, particularly those with slower HIV progression and more time with detectable viremia prior to initiation of suppressive ART, are a promising population to identify and further study functional antibodies against HIV-1.     

Characterization of a <Emphasis Type="Italic">Pinus sylvestris</Emphasis> thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.